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

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

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


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

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


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

O O O O O O O O O O O O O O O B-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

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


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

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


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

O O 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

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


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

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


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

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


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

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


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

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

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

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


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

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


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

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

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


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

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


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

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

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


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

O O B-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 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 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 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 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 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 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 .

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


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

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


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

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


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

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

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

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 cells ( left panel ) and the number of TRAP - positive cells with more than three nuclei ( right ) . Scale bar , 100 μm .

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


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

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

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


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

O O O 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 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 O O

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


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

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

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


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

O O O 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 ) male mice showing osteoclasts ( left , tdTomato fluorescence ) and FLIM image for ATP changes ( middle and right , fluorescence lifetime of EGFP ) . The FRET efficiencies of each osteoclast were plotted from the mice upon exposure to low oxygen atmosphere ( 14 % pO2 ) or ambient air ( right , n = 11 from three mice for each SpO2 ) .

O O O O O O O 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

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


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

O O O O O O O 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

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


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

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

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

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

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

O O O O 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 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 O O O O

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


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

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

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


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

O O O O 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 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

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


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

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

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


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

O O O O 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 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 B-MEASURED_VAR 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

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


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

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

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


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

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

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


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

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

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


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

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

O O O O O 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 O O

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


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

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

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

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

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

O O O O 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 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

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


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

O O O O 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 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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


I , J : In I One - way ANOVA was used to compare the expression of 9 neuronal genes between Mecp2 null primary neurons ( set to 100 ) , null neurons treated with CX546 from DIV1 to DIV4 and null neurons treated with CX546 and Nifedipine from DIV1 to DIV4 . * refers to differences between Mecp2 null untreated neurons and Mecp2 null neurons treated with CX546 ( * : p - value < 0 . 05 ; * * : p - value < 0 . 01 ; * * * : p - value < 0 . 001 ) ; # refers to differences between Mecp2 null neurons treated with CX546 and null neurons treated with both CX546 and Nifedipine ( # : p - value < 0 . 05 ; # # : p - value < 0 . 01 ) . Panel J shows the ratio between the expression level of Kcc2 and Nkcc1 ( expressed as 2 - ∆ ct ) . Box plot shows the median ( central band ) , the lower and upper quartiles ( boxes ) and the max and min values ( whiskers ) from at least 3 wells obtained from four different embryos . One - way ANOVA was used to compare the effects of the different treatments on null neurons compared to the untreated ones . * * : p - value < 0 . 01 . Sample size : n ≥ 3 wells from four different embryos .

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

I,J: In I One-way ANOVA was used to compare the expression of 9 neuronal genes between Mecp2 null primary neurons (set to 100), null neurons treated with CX546 from DIV1 to DIV4 and null neurons treated with CX546 and Nifedipine from DIV1 to DIV4. * refers to differences between Mecp2 null untreated neurons and Mecp2 null neurons treated with CX546 (*: p-value<0.05; **: p-value<0.01; ***: p-value<0.001); # refers to differences between Mecp2 null neurons treated with CX546 and null neurons treated with both CX546 and Nifedipine (#: p-value<0.05; ##: p-value<0.01). Panel J shows the ratio between the expression level of Kcc2 and Nkcc1 (expressed as 2-∆ct). Box plot shows the median (central band), the lower and upper quartiles (boxes) and the max and min values (whiskers) from at least 3 wells obtained from four different embryos. One-way ANOVA was used to compare the effects of the different treatments on null neurons compared to the untreated ones. **: p-value<0.01. Sample size: n≥3 wells from four different embryos.


B : Kaplan Mayer test shows an improved lifespan of Mecp2 null CX546 treated animals compared to Mecp2 null control animals . Sample size : wt = 11 , ko = 16 , ko treated with CX546 = 11 . Gehan - Breslow - Wilcoxon test was used to compare the two groups . * * : p - value < 0 . 01 .

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

B: Kaplan Mayer test shows an improved lifespan of Mecp2 null CX546 treated animals compared to Mecp2 null control animals. Sample size: wt=11, ko=16, ko treated with CX546=11. Gehan-Breslow-Wilcoxon test was used to compare the two groups. **: p-value<0.01.


F , G : Line graph ( F ) shows mice motor learning assessed on the accelerating rotarod during three consecutive trials . Two - way ANOVA indicated a significant effect of time ( F ( 2 , 21 ) = 5 , 490 and p = 0 , 0121 ) and genotype ( F ( 2 , 33 ) = 16 , 33 and p < 0 . 0001 ) . Bonferroni multiple comparison test highlighted a significant increase in the latency to fall between trial 1 and trial 3 only for null mice treated with CX546 . * : p - value < 0 . 05 ; * * : p - value < 0 . 01 . The scatter plot ( G ) shows latency to fall ( in seconds ) on the accelerating rotarod in the last trial . One - way ANOVA was used to compare ko mice and ko treated mice to the wt group . The Tukey ' s multiple comparison test highlighted a significant reduction in the latency to fall between wt and ko but not between wt and ko treated with Ampakine ; * * : p - value < 0 . 01 . Values are represented as average ± SEM . Sample size : n = 6 wt treated with vehicle and n = 14 ko ( 6 treated with vehicle and 8 treated with CX546 ) . Each dot represents a single animal .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,G: Line graph (F) shows mice motor learning assessed on the accelerating rotarod during three consecutive trials. Two-way ANOVA indicated a significant effect of time (F(2,21)=5,490 and p=0,0121) and genotype (F(2,33)=16,33 and p<0.0001). Bonferroni multiple comparison test highlighted a significant increase in the latency to fall between trial 1 and trial 3 only for null mice treated with CX546. *: p-value<0.05; **: p-value<0.01. The scatter plot (G) shows latency to fall (in seconds) on the accelerating rotarod in the last trial. One-way ANOVA was used to compare ko mice and ko treated mice to the wt group. The Tukey's multiple comparison test highlighted a significant reduction in the latency to fall between wt and ko but not between wt and ko treated with Ampakine; **: p-value<0.01. Values are represented as average ± SEM. Sample size: n=6 wt treated with vehicle and n=14 ko (6 treated with vehicle and 8 treated with CX546). Each dot represents a single animal.


A , B After grown in nutrient rich medium to log phase , wild - type ( WT ) cells were treated with rapamycin ( 10 μM ) or subject to nitrogen starvation ( SD - N ) for 3 h and 16 h and degradation of autophagic marker GFP - Atg8 was detected . The cycloheximide ( CHX , 100 μg / mL , ) or thiolutin ( 10 μg / mL ) was simultaneously used to block protein synthesis or gene transcription .

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

A, B After grown in nutrient rich medium to log phase, wild-type (WT) cells were treated with rapamycin (10 μM) or subject to nitrogen starvation (SD-N) for 3 h and 16 h and degradation of autophagic marker GFP-Atg8 was detected. The cycloheximide (CHX, 100 μg/mL,) or thiolutin (10 μg/mL) was simultaneously used to block protein synthesis or gene transcription.


Yeast cells were treated with thiolutin ( 10 μg / mL ) together with rapamycin or nitrogen starvation in different order for 3 h , 6h and 16 h and degradation of autophagic marker GFP - Atg8 was detected .

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Yeast cells were treated with thiolutin (10 μg/mL) together with rapamycin or nitrogen starvation in different order for 3 h, 6h and 16 h and degradation of autophagic marker GFP-Atg8 was detected.


B N - terminally GFP - tagged 50Q was checked for its autophagic degradation in indicated yeast cells by GFP processing assays after 1 , 4 and 16 h starvation . Yeast cells with ATG1 deletion were used as positive controls .

O O O O O O O O O O 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 N-terminally GFP-tagged 50Q was checked for its autophagic degradation in indicated yeast cells by GFP processing assays after 1, 4 and 16 h starvation. Yeast cells with ATG1 deletion were used as positive controls.


H Vacuole localization and degradation of endocytosis substrate GFP - Sna3 in WT , atg1 ∆ and rpb9 ∆ cells . Scale bars : 5 μm .

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H Vacuole localization and degradation of endocytosis substrate GFP-Sna3 in WT, atg1∆ and rpb9∆ cells. Scale bars: 5 μm.


I Carboxypeptidases Y ( Cpy1 ) , a vacuolar - resident hydrolases , was analyzed for its transporting to vacuoles by the secretory pathway in WT , atg1 ∆ and rpb9 ∆ cells .

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I Carboxypeptidases Y (Cpy1), a vacuolar-resident hydrolases, was analyzed for its transporting to vacuoles by the secretory pathway in WT, atg1∆ and rpb9∆ cells.


E - I Overexpression of ATG1 could partially restore autophagy in rpb9 ∆ cells . ATG1 , ATG9 , ATG13 , ATG17 and ATG5 were overexpressed in rpb9 ∆ yeast cells , GFP - 50Q was checked for its autophagic degradation by GFP processing assays after 1 , 4 and 16 h starvation .

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

E-I Overexpression of ATG1 could partially restore autophagy in rpb9∆ cells. ATG1, ATG9, ATG13, ATG17 and ATG5 were overexpressed in rpb9∆ yeast cells, GFP-50Q was checked for its autophagic degradation by GFP processing assays after 1, 4 and 16 h starvation.


B Indicated Rpb9 truncates and mutants were expressed in rpb9 ∆ cells and autophagic degradation of GFP - 50Q was checked by GFP - processing assays .

O O B-CONTROLLED_VAR 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 Indicated Rpb9 truncates and mutants were expressed in rpb9∆ cells and autophagic degradation of GFP-50Q was checked by GFP-processing assays.


E Purified GST - Gcn4 and His - Rpb9 proteins from E . coli cells were mixed and GST pulldown assays were performed .

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

E Purified GST-Gcn4 and His-Rpb9 proteins from E. coli cells were mixed and GST pulldown assays were performed.


F - G Neither overexpression of Gcn4 in rpb9 ∆ yeast cells nor overexpression of Rpb9 in gcn4 ∆ yeast cells can restore autophagy . Gcn4 or Rpb9 were overexpressed in rpb9 ∆ or gcn4 ∆ yeast cells respectively and autophagic degradation of GFP - 50Q was detected .

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

F-G Neither overexpression of Gcn4 in rpb9∆ yeast cells nor overexpression of Rpb9 in gcn4∆ yeast cells can restore autophagy. Gcn4 or Rpb9 were overexpressed in rpb9∆ or gcn4∆ yeast cells respectively and autophagic degradation of GFP-50Q was detected.


C Rpb9 orthologs from complex eukaryotic species could restore autophagy in rpb9 ∆ yeast cells . Indicated Rpb9 orthologs were expressed in rpb9 ∆ yeast cells and autophagic degradation of GFP - 50Q was detected .

O B-CONTROLLED_VAR 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 O O

C Rpb9 orthologs from complex eukaryotic species could restore autophagy in rpb9∆ yeast cells. Indicated Rpb9 orthologs were expressed in rpb9∆ yeast cells and autophagic degradation of GFP-50Q was detected.


D N - terminal Zinc finger domain and linker region of Rpb9 orthologs from different species could restore autophagy defects in rpb9 ∆ yeast cells . Indicated truncates of Rpb9 orthologs were expressed in rpb9 ∆ yeast cells and autophagic degradation of GFP - 50Q was detected .

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

D N-terminal Zinc finger domain and linker region of Rpb9 orthologs from different species could restore autophagy defects in rpb9∆ yeast cells. Indicated truncates of Rpb9 orthologs were expressed in rpb9∆ yeast cells and autophagic degradation of GFP-50Q was detected.


A . Glioma tumour micro arrays ( grade II - IV gliomas ) were stained to visualise MDGI ( red ) . Samples from the epileptic brain served as a negative control . Scale bar 100 µm .

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A. Glioma tumour micro arrays (grade II-IV gliomas) were stained to visualise MDGI (red). Samples from the epileptic brain served as a negative control. Scale bar 100 µm.


B . Overall survival of grade II and III glioma patients ( n = 76 ) was significantly better when none / low MDGI ( blue line ) was detected compared to patients with moderate / high MDGI ( red line ) expression ( P = 0 . 007 ) . The cumulative survival rates were estimated by using the Kaplan - Meier 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 O O O O O O O O O O O O

B. Overall survival of grade II and III glioma patients (n = 76) was significantly better when none/low MDGI (blue line) was detected compared to patients with moderate/high MDGI (red line) expression (P = 0.007). The cumulative survival rates were estimated by using the Kaplan-Meier method.


C . Analysis of the association of MDGI mRNA expression with glioma patient survival using the GlioVis data portal . In the TCGA GBMLGG dataset ( n = 667 ) , patients with low MDGI expression ( green line ; n = 333 , events = 101 , median = 67 , 5 ) show significantly better survival than patients with high MDGI ( red line ; n = 334 , events = 138 , median = 41 . 5 ) expression ( P = 2e - 04 ) . HR = 0 . 62 ( 0 . 48 - 0 . 8 ) . The cumulative survival rates were estimated by using the Kaplan - Meier method .

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C. Analysis of the association of MDGI mRNA expression with glioma patient survival using the GlioVis data portal. In the TCGA GBMLGG dataset (n = 667), patients with low MDGI expression (green line; n = 333, events = 101, median = 67,5) show significantly better survival than patients with high MDGI (red line; n = 334, events = 138, median = 41.5) expression (P = 2e-04). HR = 0.62 (0.48 - 0.8). The cumulative survival rates were estimated by using the Kaplan-Meier method.


D . In glioblastoma samples , highest MDGI expression was observed in the mesenchymal subtype ( M ) compared to the classical ( Cl ) or pro - neural ( PN ) ones . However , it did not reach the statistical significance . GlioVis data portal , TCGA GBM dataset , n = 156 . Pairwise t - test with corrections for multiple testing , p - values with Bonferroni correction .

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D. In glioblastoma samples, highest MDGI expression was observed in the mesenchymal subtype (M) compared to the classical (Cl) or pro-neural (PN) ones. However, it did not reach the statistical significance. GlioVis data portal, TCGA GBM dataset, n = 156. Pairwise t-test with corrections for multiple testing, p-values with Bonferroni correction.


E . MDGI expression was high in the leading edge of the tumour ( LE ) and in infiltrating tumour cells ( IT ) in the IVY _ Gap dataset ( n = 122 ) where the gene expression profile in different anatomical structures of glioblastomas was analysed . LE , leading edge ; IT , infiltrating tumour cells ; CT , cellular tumour ( cells within the tumour mass ) ; PC , pseudopalisading cells ; MP , microvascular proliferation . Numbers in the graph depict the P values : Pairwise t - test with corrections for multiple testing , p - values with Bonferroni correction .

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

E. MDGI expression was high in the leading edge of the tumour (LE) and in infiltrating tumour cells (IT) in the IVY_Gap dataset (n = 122) where the gene expression profile in different anatomical structures of glioblastomas was analysed. LE, leading edge; IT, infiltrating tumour cells; CT, cellular tumour (cells within the tumour mass); PC, pseudopalisading cells; MP, microvascular proliferation. Numbers in the graph depict the P values: Pairwise t-test with corrections for multiple testing, p-values with Bonferroni correction.


F . Western blot analysis shows MDGI expression in human glioma cells including patient - derived spheroids ( S24 , ZH161 , ZH305 , BT3 , BT11 , BT12 , BT13 ) , patient - derived adherent cells ( BT5 and BT5R ) , and long - term cell lines ( U87MG , LN229 , LN308 ) . Numbers above the lanes show the relative expression of MDGI compared to the levels of β - tubulin that served as loading control in each cell line . Representative Western blot image of 3 separate experiments is shown .

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F. Western blot analysis shows MDGI expression in human glioma cells including patient-derived spheroids (S24, ZH161, ZH305, BT3, BT11, BT12, BT13), patient-derived adherent cells (BT5 and BT5R), and long-term cell lines (U87MG, LN229, LN308). Numbers above the lanes show the relative expression of MDGI compared to the levels of β-tubulin that served as loading control in each cell line. Representative Western blot image of 3 separate experiments is shown.


G . High MDGI and HIF - 1α expression in the patient - derived glioblastoma spheroids decreased significantly when cells were cultured in medium containing 10 % of serum ( FBS ) for seven days . Numbers show the relative expression of MDGI and HIF - 1α compared to the levels of β - tubulin . Expression in the serum - free medium was set as 1 . Representative Western blot image of 2 separate experiments is shown .

O O O B-MEASURED_VAR 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-MEASURED_VAR 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

G. High MDGI and HIF-1α expression in the patient-derived glioblastoma spheroids decreased significantly when cells were cultured in medium containing 10 % of serum (FBS) for seven days. Numbers show the relative expression of MDGI and HIF-1α compared to the levels of β-tubulin. Expression in the serum-free medium was set as 1. Representative Western blot image of 2 separate experiments is shown.


H . MDGI expression in adherent glioma cell lines and in glioma cell spheroids after 24h culture under hypoxia ( 1 % O2 ) or normoxia ( 21 % O2 ) . Numbers show the relative MDGI expression compared to the levels of β - tubulin . Expression under normoxia was set as 1 . Representative Western blot image of 3 separate experiments is shown .

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 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

H. MDGI expression in adherent glioma cell lines and in glioma cell spheroids after 24h culture under hypoxia (1 % O2) or normoxia (21 % O2). Numbers show the relative MDGI expression compared to the levels of β-tubulin. Expression under normoxia was set as 1. Representative Western blot image of 3 separate experiments is shown.


A , B Representative whole coronal section micrographs of murine brain injected with GFP ( A ) or MDGI - GFP ( B ) expressing U87MG glioma cells . Xenografted human cells were visualised by using anti - human vimentin ( hVim ) antibodies ( red ) . Nuclei were visualised by using DAPI ( white ) . Scale bar : 1 mm .

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A, B Representative whole coronal section micrographs of murine brain injected with GFP (A) or MDGI-GFP (B) expressing U87MG glioma cells. Xenografted human cells were visualised by using anti-human vimentin (hVim) antibodies (red). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm.


Micrographs of consecutive ( separating distance : 9 µm ) brain sections stained for human glioma cells using antibodies specific for human vimentin ( hVim red in C and D ) , Dashed lines separate the primary tumour mass and the normal brain . Nuclei were visualised by using DAPI ( blue ) . Arrows in D point to the angiotropic tumours . Scale bar : 50 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 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

Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for human vimentin (hVim red in C and D), Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). Arrows in D point to the angiotropic tumours. Scale bar: 50 µm.


Micrographs of consecutive ( separating distance : 9 µm ) brain sections stained for human glioma cells using antibodies specific for MDGI ( green in E and F ) , and CD31 ( red in E and F ) . Dashed lines separate the primary tumour mass and the normal brain . Nuclei were visualised by using DAPI ( blue ) . Arrows in F point to the MDGI - expressing angiotropic tumour cells . Scale bar : 50 µm .

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 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

Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for MDGI (green in E and F), and CD31 (red in E and F). Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). Arrows in F point to the MDGI-expressing angiotropic tumour cells. Scale bar: 50 µm.


G . Quantification of the number of U87MG secondary tumours ( diameter > 300 µm ) detected in the whole brain ( GFP : n = 5 , MDGI - GFP : n = 9 ) . Data are represented as mean ± SEM . * * P < 0 . 01 , two - tailed , nonparametric Mann - Whitney ' s U - test .

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

G. Quantification of the number of U87MG secondary tumours (diameter >300 µm) detected in the whole brain (GFP: n = 5, MDGI-GFP: n = 9). Data are represented as mean ± SEM. **P < 0.01, two-tailed, nonparametric Mann-Whitney's U-test.


H . Quantification of the number of invasive U87MG cells that grew next to brain blood vessels ( angiotropic co - opting tumour cells , diameter < 300 µm ) ( GFP : n = 5 , MDGI - GFP : n = 9 ) . Data are represented as mean ± SEM . * * * * P < 0 . 0001 , two - tailed , nonparametric Mann - Whitney ' s U - test .

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

H. Quantification of the number of invasive U87MG cells that grew next to brain blood vessels (angiotropic co-opting tumour cells, diameter <300 µm) (GFP: n = 5, MDGI-GFP: n = 9). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.


I , J Representative micrographs of whole murine brain coronal sections injected with GFP ( I ) or GFP - MDGI ( J ) expressing LN229 glioma cells . Xenografted human cells were visualised by using anti - human vimentin ( hVim ) antibodies ( red ) . Nuclei were visualised by using DAPI ( white ) . Scale bar : 1 mm .

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

I, J Representative micrographs of whole murine brain coronal sections injected with GFP (I) or GFP-MDGI (J) expressing LN229 glioma cells. Xenografted human cells were visualised by using anti-human vimentin (hVim) antibodies (red). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm.


Micrographs of consecutive ( separating distance : 9 µm ) brain sections stained for human glioma cells using antibodies specific for human vimentin ( hVim red in K and L ) Dashed lines separate the primary tumour mass and the normal brain . Nuclei were visualised by using DAPI ( blue ) . ) . Arrows in L point to the angiotropic tumours . Scale bar : 50 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 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

Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for human vimentin (hVim red in K and L) Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). ). Arrows in L point to the angiotropic tumours. Scale bar: 50 µm.


Micrographs of consecutive ( separating distance : 9 µm ) brain sections stained for human glioma cells using antibodies specific for MDGI ( green in M and N ) , and CD31 ( red in M and N ) . Dashed lines separate the primary tumour mass and the normal brain . Nuclei were visualised by using DAPI ( blue ) . ) . Arrows in N point to the MDGI - expressing angiotropic tumour cells . Scale bar : 50 µm .

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 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

Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for MDGI (green in M and N), and CD31 (red in M and N). Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). ). Arrows in N point to the MDGI-expressing angiotropic tumour cells. Scale bar: 50 µm.


O . Quantification of the number of invasive LN229 cells detected in the brain ( GFP : n = 10 , MDGI - GFP : n = 10 ) . Data are represented as mean ± SEM . * * * * P < 0 . 0001 , two - tailed , nonparametric Mann - Whitney ' s U - test .

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O. Quantification of the number of invasive LN229 cells detected in the brain (GFP: n = 10, MDGI-GFP: n = 10). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.


P . Quantification of the number of invasive LN229 cells that grew next to brain blood vessels ( angiotropic co - opting tumour cells , diameter < 300 µm ) detected in the brain ( GFP : n = 10 , MDGI - GFP : n = 10 ) . Data are represented as mean ± SEM . Data are represented as mean ± SEM . * * * * P < 0 . 0001 , two - tailed , nonparametric Mann - Whitney ' s U - test .

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P. Quantification of the number of invasive LN229 cells that grew next to brain blood vessels (angiotropic co-opting tumour cells, diameter <300 µm) detected in the brain (GFP: n = 10, MDGI-GFP: n = 10). Data are represented as mean ± SEM. Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.


A . Transmitted light micrographs of MDGI - silenced ( shMDGI1 and shMDGI2 ) and control ( Scr ) patient - derived BT12 and BT13 spheroids 11 days after gene silencing . Representative images of at least 3 independent experiments are shown . Scale bar : 200 μm .

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A. Transmitted light micrographs of MDGI-silenced (shMDGI1 and shMDGI2) and control (Scr) patient-derived BT12 and BT13 spheroids 11 days after gene silencing. Representative images of at least 3 independent experiments are shown. Scale bar: 200 μm.


B . Methylcellulose assay demonstrating the ability for self - renewal of control ( Scr ) and MDGI - silenced ( shMDGI1 and shMDGI2 ) patient - derived BT12 and BT13 cells after three weeks of culture . Representative images of at least 3 independent experiments are shown . Scale bar : 200 μm .

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B. Methylcellulose assay demonstrating the ability for self-renewal of control (Scr) and MDGI-silenced (shMDGI1 and shMDGI2) patient-derived BT12 and BT13 cells after three weeks of culture. Representative images of at least 3 independent experiments are shown. Scale bar: 200 μm.


C . Quantification of the colony size in methylcellulose of MDGI - silenced ( sh1 and sh2 ) cells relative to the controls ( Scr ) that is set as 100 ( n = 3 ) . Data are represented as mean ± SD . * * * P < 0 . 001 , two - tailed , nonparametric Mann - Whitney ' s U - test .

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C. Quantification of the colony size in methylcellulose of MDGI-silenced (sh1 and sh2) cells relative to the controls (Scr) that is set as 100 (n = 3). Data are represented as mean ± SD. ***P < 0.001, two-tailed, nonparametric Mann-Whitney's U-test.


D . MTT - proliferation assay of control ( Scr ) and MDGI - silenced ( shMDGI1 ) BT12 and BT13 cells . The values of each cell line were normalized to the values at day 1 marked with a dashed line ( n = 3 ) . Data are represented as mean ± SD . * * * P < 0 . 001 , two - tailed , nonparametric Mann - Whitney ' s U - test .

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D. MTT-proliferation assay of control (Scr) and MDGI-silenced (shMDGI1) BT12 and BT13 cells. The values of each cell line were normalized to the values at day 1 marked with a dashed line (n = 3). Data are represented as mean ± SD. ***P < 0.001, two-tailed, nonparametric Mann-Whitney's U-test.


E , F Representative whole coronal section micrographs of murine brain injected intracranially with control ( BT12 Scr , n = 5 ) and MDGI - silenced ( BT12 sh1 , n = 5 ) BT12 cells . After four weeks animals were sacrificed , the brains were excised and prepared for immunohistochemistry . Control ( Scr ) cells grew very similarly to the parental BT12 cells forming a tumour mass with invasive tumour cells ( single cell invasion ) that eventually co - opted existing blood vessels ( angiotropism ) and formed secondary tumours ( upper panels ) . No tumour growth was observed when MDGI - silenced cells were implanted ( lower panels ) . Glioma cells were visualised by using anti - human vimentin ( hVim red ) and blood vessels using anti - podocalyxin antibodies ( green ) . Nuclei were visualised by using DAPI ( white ) . Scale bar : 1 mm ( E ) and 50 µm ( F ) .

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E, F Representative whole coronal section micrographs of murine brain injected intracranially with control (BT12 Scr, n = 5) and MDGI-silenced (BT12 sh1, n = 5) BT12 cells. After four weeks animals were sacrificed, the brains were excised and prepared for immunohistochemistry. Control (Scr) cells grew very similarly to the parental BT12 cells forming a tumour mass with invasive tumour cells (single cell invasion) that eventually co-opted existing blood vessels (angiotropism) and formed secondary tumours (upper panels). No tumour growth was observed when MDGI-silenced cells were implanted (lower panels). Glioma cells were visualised by using anti-human vimentin (hVim red) and blood vessels using anti-podocalyxin antibodies (green). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm (E) and 50 µm (F).


A . Binding of Annexin V Alexa Fluor 488 to control ( Scr ) and MDGI - silenced ( shMDGI1 ) BT12 cells was measured by flow cytometric analyses five days after silencing . The analysis was performed by gating the cells into two populations : P1 ( small , granular ) and P2 ( normal - sized cells ) ( 30 000 analysed events / cell line , n = 3 ) . B . Geometric mean fluorescence ( FL - 1 ) relative to the controls ( Scr ) that was set as 1 in P1 and P2 in MDGI - silenced ( shMDGI1 ) cells . Data are represented as mean ± SD . * P < 0 . 05 , two - tailed , nonparametric Mann - Whitney ' s U - test .

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A. Binding of Annexin V Alexa Fluor 488 to control (Scr) and MDGI-silenced (shMDGI1) BT12 cells was measured by flow cytometric analyses five days after silencing. The analysis was performed by gating the cells into two populations: P1 (small, granular) and P2 (normal-sized cells) (30 000 analysed events/cell line, n = 3). B. Geometric mean fluorescence (FL-1) relative to the controls (Scr) that was set as 1 in P1 and P2 in MDGI-silenced (shMDGI1) cells. Data are represented as mean ± SD. *P < 0.05, two-tailed, nonparametric Mann-Whitney's U-test.


C , D Representative micrographs of non - treated control BT12 cells ( C ) and cells treated with 2 mM LLOMe for 4 h ( D ) before staining with the anti - galectin - 1 antibodies ( LGALS1 ) ( green ) . Nuclei were visualised by using DAPI ( blue ) . Scale bar : 10 µm .

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C, D Representative micrographs of non-treated control BT12 cells (C) and cells treated with 2 mM LLOMe for 4 h (D) before staining with the anti-galectin-1 antibodies (LGALS1) (green). Nuclei were visualised by using DAPI (blue). Scale bar: 10 µm.


E , F Representative micrographs of LGALS1 - stained ( green ) BT12 control ( E ) and MDGI - silenced cells ( F ) six days after silencing . Nuclei were visualised by using DAPI ( blue ) . Scale bar : 10 µm .

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E, F Representative micrographs of LGALS1-stained (green) BT12 control (E) and MDGI-silenced cells (F) six days after silencing. Nuclei were visualised by using DAPI (blue). Scale bar: 10 µm.


G , H Representative micrographs of LGALS1 - stained ( green ) BT13 control ( G ) and MDGI - silenced cells ( H ) six days after silencing . Nuclei were visualised by using DAPI ( blue ) . Scale bar : 10 µm .

G, H Representative micrographs of LGALS1-stained (green) BT13 control (G) and MDGI-silenced cells (H) six days after silencing. Nuclei were visualised by using DAPI (blue). Scale bar: 10 µm.