( a ) Growth of L . monocytogenes in S2 ( S2 ) or S2 expressing PGRP - LE ( S2 - LE ) transfected with double - stranded ( RNAi ) specific for Atg5 , Rel or Dif dl ( Dif - dl ) infected for 1 . 5 h with wild - type or Δhly L . monocytogenes , followed by 6 h of incubation in medium containing CuSO4 gentamicin , quantified by plate of colony - forming units . * , P 0 . 001 ( t - ) . Data are representative of two independent experiments ( error bars , s . d . ) .

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(a) Growth of L. monocytogenes in S2 cells (S2) or S2 cells expressing PGRP-LE (S2-LE) transfected with double-stranded RNA (RNAi) specific for Atg5, Rel or Dif and dl (Dif-dl) and infected for 1.5 h with wild-type or Δhly L. monocytogenes, followed by 6 h of incubation in medium containing CuSO4 and gentamicin, quantified by plate assay of colony-forming units. *, P 0.001 (t-test). Data are representative of two independent experiments (error bars, s.d.).


( b , c ) confocal microscopy of localization of PGRP - LE together with wild - type L . monocytogenes in S2 engineered to express YFP - PGRP - LE ( YFP - LE ) infected for 0 . 5 h with wild - type L . monocytogenes ( b ) or Δhly L . monocytogenes ( c ) , followed by 1 h of incubation in gentamicin - containing medium , then DAPI . Merge , YFP - PGRP - LE ( green ) DAPI ( magenta ) . Filled arrowheads indicate L . monocytogenes ; open arrowhead indicates accumulation of YFP - PGRP - LE around . Scale bars , 5 μm . Data are representative of three experiments .

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(b,c) Fluorescence confocal microscopy of the localization of PGRP-LE together with wild-type L. monocytogenes in S2 cells engineered to express YFP-PGRP-LE (YFP-LE) and infected for 0.5 h with wild-type L. monocytogenes (b) or Δhly L. monocytogenes (c), followed by 1 h of incubation in gentamicin-containing medium, then DAPI staining. Merge, YFP-PGRP-LE (green) and DAPI (magenta). Filled arrowheads indicate L. monocytogenes; open arrowhead indicates accumulation of YFP-PGRP-LE around the bacteria. Scale bars, 5 μm. Data are representative of three experiments.


( a ) Dot - or ring - shaped GFP - LC3 ( LC3 dot ) signals in S2 expressing both PGRP - LE GFP - LC3 under control of an actin promoter ( PGRP - LE GFP - LC3 ) or GFP - LC3 alone ( GFP - LC3 ) after infection with wild - type or Δhly L . monocytogenes or after 1 . 5 h of incubation with 5 μM rapamycin ( rap ) . - , no infection .

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(a) Dot- or ring-shaped GFP-LC3 (LC3 dot) signals in S2 cells expressing both PGRP-LE and GFP-LC3 under the control of an actin promoter (PGRP-LE GFP-LC3) or GFP-LC3 alone (GFP-LC3) after infection with wild-type or Δhly L. monocytogenes or after 1.5 h of incubation with 5 μM rapamycin (rap). -, no infection.


( b ) Colocalization of GFP - LC3 dots wild - type L . monocytogenes in S2 , quantified by confocal microscopy .

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(b) Colocalization of GFP-LC3 dots and wild-type L. monocytogenes in S2 cells, quantified by confocal microscopy.


( c ) Confocal microscopy of S2 expressing PGRP - LE GFP - LC3 ( green ) infected with wild - type L . monocytogenes stained with DAPI ( magenta ) . Arrow indicates colocalization of GFP - LC3 L . monocytogenes .

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(c) Confocal microscopy of S2 cells expressing PGRP-LE and GFP-LC3 (green) infected with wild-type L. monocytogenes and stained with DAPI (magenta). Arrow indicates colocalization of GFP-LC3 and L. monocytogenes.


( d ) Immunoblot of lysates of S2 left untreated ( - ) or infected with wild - type L . monocytogenes or treated with 5 μM rapamycin .

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(d) Immunoblot analysis of lysates of S2 cells left untreated (-) or infected with wild-type L. monocytogenes or treated with 5 μM rapamycin.


( e - i ) Ultrastructural of S2 expressing PGRP - LE GFP - LC3 infected with wild - type L . monocytogenes . ( e , f ) microscopy ( e ) electron microscopy ( f ) of expressing GFP - LC3 ( green ) stained with DAPI ( magenta ) . ( g ) Enlargement of a - containing vacuole from f . ( h , i ) Enlargement of fields outlined in g . Arrows indicate double - structure that surrounds ; arrowheads indicate an endoplasmic reticulum - like .

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(e-i) Ultrastructural analysis of S2 cells expressing PGRP-LE and GFP-LC3 and infected with wild-type L. monocytogenes. (e,f) Fluorescence microscopy (e) and electron microscopy (f) of cells expressing GFP-LC3 (green) and stained with DAPI (magenta). (g) Enlargement of a bacteria-containing vacuole from f. (h,i) Enlargement of the fields outlined in g. Arrows indicate double-membrane structure that surrounds the bacteria; arrowheads indicate an endoplasmic reticulum-like membrane.


( j ) Confocal microscopy of hemocytes infected with wild - type L . monocytogenes . Green , GFP - LC3 ; magenta , DAPI . Arrow indicates colocalization of GFP - LC3 L . monocytogenes .

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(j) Confocal microscopy of hemocytes infected with wild-type L. monocytogenes. Green, GFP-LC3; magenta, DAPI. Arrow indicates colocalization of GFP-LC3 and L. monocytogenes.


( k ) Dot - or ring - shaped GFP - LC3 signals in ex vivo - cultured hemocytes expressing GFP - LC3 ( infection treatment , horizontal axis ) .

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(k) Dot- or ring-shaped GFP-LC3 signals in ex vivo-cultured hemocytes expressing GFP-LC3 (infection and treatment, horizontal axis).


( l ) Immunoblot of lysates of hemocytes from third instar larvae cultured ex vivo infected with L . monocytogenes or treated with rapamycin . Arrowhead indicates processed form of GFP - LC3 .

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(l) Immunoblot analysis of lysates of hemocytes from third instar larvae cultured ex vivo and infected with L. monocytogenes or treated with rapamycin. Arrowhead indicates processed form of GFP-LC3.


( m ) GFP - LC3 dots in S2 expressing PGRP - LE GFP - LC3 treated with RNAi ( below graph ) infected for 0 . 5 h with L . monocytogenes then incubated for 1 h in gentamicin - containing medium ; dots quantified by confocal microscopy . Scale bars , 5 μm ( c , j ) , 1 μm ( e , f ) or 500 nm ( g ) . * , P 0 . 001 ( t - ) . Data are representative of three ( a , k , m ) , two ( b , e - i ) , six ( c ) , four ( d ) or five ( j , l ) experiments ( error bars , s . d . of triplicate measurements ( a , b , l ) or at least triplicate measurements ( m ) ) .

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(m) GFP-LC3 dots in S2 cells expressing PGRP-LE and GFP-LC3 and treated with RNAi (below graph) and infected for 0.5 h with L. monocytogenes then incubated for 1 h in gentamicin-containing medium; dots quantified by confocal microscopy. Scale bars, 5 μm (c,j), 1 μm (e,f) or 500 nm (g). *, P 0.001 (t-test). Data are representative of three (a,k,m), two (b,e-i), six (c), four (d) or five (j,l) experiments (error bars, s.d. of triplicate measurements (a,b,l) or at least triplicate measurements (m)).


( a ) GFP - LC3 dots in S2 expressing GFP - LC3 alone or PGRP - LE GFP - LC3 , treated for 2 h with TCT ( 100 nM ) , highly purified DAP - type PGN from L . plantarum ( DAP ; 100 μg / ml ) or lysine - type PGN from S . epidermidis ( Lys ) .

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(a) GFP-LC3 dots in S2 cells expressing GFP-LC3 alone or PGRP-LE and GFP-LC3, treated for 2 h with TCT (100 nM), highly purified DAP-type PGN from L. plantarum (DAP; 100 μg/ml) or lysine-type PGN from S. epidermidis (Lys).


( b ) Dot - or ring - shaped GFP - LC3 signals in hemocytes after infection with wild - type or Δhly L . monocytogenes or treatment with rapamycin ( 5 μM ) or with TCT , DAP - type PGN or lysine - type PGN . * , P 0 . 001 ( t - ) . Data are representative of two independent experiments ( error bars , s . d . ) .

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(b) Dot- or ring-shaped GFP-LC3 signals in hemocytes after infection with wild-type or Δhly L. monocytogenes or treatment with rapamycin (5 μM) or with TCT, DAP-type PGN or lysine-type PGN. *, P 0.001 (t-test). Data are representative of two independent experiments (error bars, s.d.).


Left : changes of IRDye700 labelled dsDNA ( at 125 nM ) when incubated at increasing ID2 ( I + D2 , IUb + D2Ub , I + D2Ub ) or ubiquitinated FANCD2 ( D2Ub ) concentrations ( ranging from 1 . 3 nM to 5 . 9 µM ) . Measurement of enhancement for each ID2 was conducted for two separately prepared complexes ( two technical repeats ) data points for each - combination were used in fitting of a one - site binding model . Right : Bar graph showing mean apparent Kd values calculated from model fitting . Error bars : Asymmetric 95 % confidence intervals from non - linear regression ( 23 - 24 data points each ) .

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Left: Fluorescence changes of IRDye700 labelled dsDNA (at 125 nM) when incubated at increasing ID2 (I+D2, IUb+D2Ub, I+D2Ub) or ubiquitinated FANCD2 (D2Ub) concentrations (ranging from 1.3 nM to 5.9 µM). Measurement of fluorescence enhancement for each ID2 complex was conducted for two separately prepared complexes (two technical repeats) and all data points for each protein-combination were used in fitting of a one-site binding model. Right: Bar graph showing mean apparent Kd values calculated from model fitting. Error bars: Asymmetric 95% confidence intervals from non-linear regression (23-24 data points each).


Assessment of - interactions using Electro - mobility shift assays ( EMSA ) . IRDye700 labelled dsDNA ( at 2 nM ) was incubated with indicated amounts of non / single / double - ubiquitinated ID2 ( His6 - TEV - V5 - FANCI FLAG - FANCD2 ) complexes ( I + D2 , IUb + D2Ub , I + D2Ub ) or ubiquitinated FLAG - FANCD2 ( D2Ub ) . Mixes were ran on non - denaturing gels , resolved free - - bound bands were visualized using an infrared scanner . EMSA gels of ID2 complexes are representative of 3 replicate experiments .

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Assessment of protein-DNA interactions using Electro-mobility shift assays (EMSA). IRDye700 labelled dsDNA (at 2 nM) was incubated with indicated amounts of non/single/double-ubiquitinated ID2 (His6-TEV-V5-FANCI and FLAG-FANCD2) protein complexes (I+D2, IUb+D2Ub, I+D2Ub) or ubiquitinated FLAG-FANCD2 (D2Ub). Mixes were ran on non-denaturing gels, and the resolved free- and protein-bound DNA bands were visualized using an infrared scanner. EMSA gels of ID2 complexes are representative of 3 replicate experiments.


Left : CryoEM 2D classes of ID2Ub IUbD2Ub - dsDNA . Right : 3D reconstructions of ID2Ub ( EMD - 10843 ) IUbD2Ub - ( EMD - 10844 ) . Both structures exhibit a torus - like shape .

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Left: CryoEM 2D classes of ID2Ub and IUbD2Ub-dsDNA. Right: 3D reconstructions of ID2Ub (EMD-10843) and IUbD2Ub-DNA (EMD-10844). Both structures exhibit a torus-like shape.


Both wild - type ( IWT ) R1285Q mutant ( IR1285Q ) FANCI can be efficiently ubiquitinated in presence of . Reactions were performed at 2 μM FANCI substrate for 60 min in absence or presence of 4 µM ssDNA .

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Both wild-type (IWT) and R1285Q mutant (IR1285Q) FANCI can be efficiently ubiquitinated in the presence of DNA. Reactions were performed at 2 μM FANCI substrate for 60 min in the absence or presence of 4 µM ssDNA.


Both wild - type ( IWT ) R1285Q mutant ( IR1285Q ) FANCI efficiently associate with FANCD2 . changes occurring when RED - tris - NTA - labelled FANCI ( IWT or IR1285Q ; both at 60 nM ) is incubated at increasing concentrations of FANCD2 ( ranging from 2 . 48 nM to 5 . 08 µM ) . Titrations were conducted twice ( two technical replicates ) data points for each - combination were used in fitting of a one - site binding model . Apparent Kd values ( mean ± ) derived from fitting of a one - site binding model to 24 data points of two technical replicates are shown .

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Both wild-type (IWT) and R1285Q mutant (IR1285Q) FANCI efficiently associate with FANCD2. Fluorescence changes occurring when RED-tris-NTA-labelled FANCI (IWT or IR1285Q; both at 60 nM) is incubated at increasing concentrations of FANCD2 (ranging from 2.48 nM to 5.08 µM). Titrations were conducted twice (two technical replicates) and all data points for each protein-combination were used in fitting of a one-site binding model. Apparent Kd values (mean ± SEM) derived from fitting of a one-site binding model to the 24 data points of the two technical replicates are shown.


FANCD2 within an IR1285QD2 is resistant to ubiquitination . Reactions were performed at 4 μM ID2 substrate 16 μM dsDNA . Progress of FANCD2 FANCI ubiquitination was monitored by western blotting following SDS - PAGE .

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FANCD2 within an IR1285QD2 complex is resistant to ubiquitination. Reactions were performed at 4 μM ID2 substrate and 16 μM dsDNA. Progress of FANCD2 and FANCI ubiquitination was monitored by western blotting following SDS-PAGE.


FANCD2 ubiquitination cannot robustly enhance ID2 - binding when complexed with IR1285Q , as determined by PIFE . Left : changes when IRD700 - labelled dsDNA ( at 125 nM ) is incubated at increasing concentrations of IR1285Q + D2 or IR1285Q + D2Ub ( concentrations ranging from 1 . 54 nM to 3 . 8 µM ) . Measurement of enhancement for each combination was conducted for two separately prepared complexes ( two technical repeats ) data points for each - combination were used in fitting of a one - site binding model . Right : Bar graph showing mean apparent Kd values calculated from one - site binding model . Error bars : Asymmetric 95 % confidence intervals from non - linear regression ( 22 data points each ) . IWT + D2 IWT + D2Ub previously calculated curves corresponding Kd values ( from data points shown in Figure 1A ) are also shown for comparison .

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FANCD2 ubiquitination cannot robustly enhance ID2-DNA binding when complexed with IR1285Q, as determined by PIFE. Left: Fluorescence changes when IRD700-labelled dsDNA (at 125 nM) is incubated at increasing concentrations of IR1285Q +D2 or IR1285Q +D2Ub (complex concentrations ranging from 1.54 nM to 3.8 µM). Measurement of fluorescence enhancement for each protein combination was conducted for two separately prepared complexes (two technical repeats) and all data points for each protein-combination were used in fitting of a one-site binding model. Right: Bar graph showing mean apparent Kd values calculated from the one-site binding model. Error bars: Asymmetric 95% confidence intervals from non-linear regression (22 data points each). IWT + D2 and IWT + D2Ub previously calculated curves and corresponding Kd values (from data points shown in Figure 1A) are also shown for comparison.


FANCD2 ubiquitination cannot robustly enhance ID2 - binding when complexed with IR1285Q , as determined by EMSAs . IRDye700 - labelled dsDNA ( at 2 nM ) was incubated with indicated amounts of ID2 ( His6 - FANCI or His6 - FANCIR1285Q mixed with non - ubiquitinated or ubiquitinated FLAG - FANCD2 . Mixes were ran on non - denaturing gels , resolved free - - bound bands were visualized using an infrared scanner . Gels shown are representative of two replicate experiments .

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FANCD2 ubiquitination cannot robustly enhance ID2-DNA binding when complexed with IR1285Q, as determined by EMSAs. IRDye700-labelled dsDNA (at 2 nM) was incubated with indicated amounts of ID2 (His6-FANCI or His6-FANCIR1285Q mixed with non-ubiquitinated or ubiquitinated FLAG-FANCD2. Mixes were ran on non-denaturing gels, and the resolved free- and protein-bound DNA bands were visualized using an infrared scanner. Gels shown are representative of two replicate experiments.


USP1 - UAF1 can efficiently deubiquitinate D2Ub in presence or absence of FANCI ( I ) , but not in presence of ubiquitinated FANCI ( IUb ) . Ubiquitinated FANCD2 ( D2Ub ) was mixed with a 50 base pair dsDNA either His6 - TEV - V5 - FANCI ( I ) , ubiquitinated His6 - TEV - V5 - FANCI ( IUb ) or no ; - mixes were subsequently incubated with USP1 - UAF1 ( at 25 nM or 100 nM ) for indicated time periods . Deubiquitination of D2Ub IUb was assessed , following SDS - PAGE , by Coomassie of gels , as well as by western blotting of transferred blots with a specific FANCD2 antibody .

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USP1-UAF1 can efficiently deubiquitinate D2Ub in the presence or absence of FANCI (I), but not in the presence of ubiquitinated FANCI (IUb). Ubiquitinated FANCD2 (D2Ub) was mixed with a 50 base pair dsDNA and either His6-TEV-V5-FANCI (I), ubiquitinated His6-TEV-V5-FANCI (IUb) or no protein; protein-DNA mixes were subsequently incubated with USP1-UAF1 (at 25 nM or 100 nM) for indicated time periods. Deubiquitination of D2Ub and IUb was assessed, following SDS-PAGE, by Coomassie staining of the gels, as well as by western blotting of transferred blots with a specific FANCD2 antibody.


USP7 USP2 can deubiquitinate D2Ub , but their activity towards D2Ub is greatly reduced in presence of FANCI ( I ) or ubiquitinated FANCI ( IUb ) . Reactions were set - up as in A , but with 100 nM USP7 or USP2 . Deubiquitination was assessed , following SDS - PAGE , by Coomassie .

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USP7 and USP2 can deubiquitinate D2Ub, but their activity towards D2Ub is greatly reduced in the presence of FANCI (I) or ubiquitinated FANCI (IUb). Reactions were set-up as in A, but with 100 nM USP7 or USP2. Deubiquitination was assessed, following SDS-PAGE, by Coomassie staining.


Time course of FANCI ubiquitination with various ubiquitin mutants corresponding sensitivity / resistance to USP1 - UAF1 mediated deubiquitination of resulting products . Deubiquitination reactions in presence or absence of USP1 - UAF1 ( 100 nM ) incubated for 30 minutes .

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Time course of FANCI ubiquitination with various ubiquitin mutants and corresponding sensitivity/resistance to USP1-UAF1 mediated deubiquitination of resulting products. Deubiquitination reactions in the presence or absence of USP1-UAF1 (100 nM) incubated for 30 minutes.


Time course of USP1 - UAF1 mediated deubiquitination of IUbD2Ub - complexes consisting of D2Ub a 50 base pair dsDNA IUb produced with various ubiquitin mutants or wild - type ( WT ) ubiquitin . Progress of deubiquitination reaction was assessed following SDS - PAGE , by both Coomassie of gels , as well as by western blotting of transferred blots with a specific FANCD2 antibody . FANCI ubiquitination with indicated ubiquitin - mutants ( or WT ) subsequent deubiquitination of resulting IUbD2Ub - complexes were conducted twice ; residual FANCD2 ubiquitination , calculated from FANCD2 blots for each time - point , was plotted for each type of ubiquitin in , corresponding deubiquitination progress curves were fitted using a one - phase decay model .

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Time course of USP1-UAF1 mediated deubiquitination of IUbD2Ub-DNA complexes consisting of D2Ub a 50 base pair dsDNA and IUb produced with various ubiquitin mutants or wild-type (WT) ubiquitin. Progress of deubiquitination reaction was assessed following SDS-PAGE, by both Coomassie staining of the gels, as well as by western blotting of transferred blots with a specific FANCD2 antibody. FANCI ubiquitination with indicated ubiquitin-mutants (or WT) and subsequent deubiquitination of resulting IUbD2Ub-DNA complexes were conducted twice; the residual FANCD2 ubiquitination, calculated from the FANCD2 blots for each time-point, was plotted for each type of ubiquitin in the protein complex, and corresponding deubiquitination progress curves were fitted using a one-phase decay model.


A . Viability of GICs treated with α - AR antagonists prazosin , ARC 239 , doxazosin , BMY 7378 , terazosin . GICs were treated with antagonists or corresponding vehicles for 72 h , viability was assayed using WST - 1 . * P < 0 . 05 , n = 4 , two - sided Mann - Whitney U - .

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A. Viability analysis of GICs treated with the α-AR antagonists prazosin, ARC 239, doxazosin, BMY 7378, and terazosin. GICs were treated with the antagonists or corresponding vehicles for 72 h, and viability was assayed using WST-1. *P<0.05, n=4, two-sided Mann-Whitney U-test.


B . of GIC survival using trypan blue exclusion after 24 h 72 h of treatment with prazosin . * P = 0 . 0286 , n = 4 , two - sided Mann - Whitney U - .

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B. Quantification of GIC survival using trypan blue exclusion after 24 h and 72 h of treatment with prazosin. *P=0.0286, n=4, two-sided Mann-Whitney U-test.


C . Viability of - derived GICs ( TG1 , TG16 , GBM5 , GBM44 ) NSCs ( NSC24 , NSC25 , NSC5031 , NSC8853 ) treated with prazosin for 72 h . * P = 0 . 0286 , n = 4 , two - sided Mann - Whitney U - .

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C. Viability analysis of patient-derived GICs (TG1, TG16, GBM5, GBM44) and NSCs (NSC24, NSC25, NSC5031, NSC8853) treated with prazosin for 72 h. *P=0.0286, n=4, two-sided Mann-Whitney U-test.


D . of sphere - forming capabilities of GICs using extreme limiting dilution . were seeded in presence of vehicle or 30 µM prazosin ( PRZ ) . Sphere formation was scored 10 days post - seeding . Frequency of sphere - forming : Control = 1 / 3 . 88 ( lower 8 . 61 , upper 1 . 95 ) ; prazosin 1 / 248 ( lower 1003 , upper 62 ) , n = 12 , p = 1 . 13 10 - 10 . Overall for difference in number between groups .

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D. Analysis of the sphere-forming capabilities of GICs using the extreme limiting dilution assay. Cells were seeded in presence of vehicle or 30 µM prazosin (PRZ). Sphere formation was scored 10 days post-seeding. Frequency of sphere-forming cells: Control=1/3.88 (lower 8.61, upper 1.95); prazosin 1/248 (lower 1003, upper 62), n=12, p= 1.13 10-10. Overall test for difference in stem cell number between groups.


E . Viability of GICs treated with prazosin after sorting according to their expression of EGFR , neural markers CD15 CD133 . Prazosin inhibits viability regardless of CD133 or CD15 expression . * P = 0 . 0286 , n = 4 , two - sided Mann - Whitney U - .

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E. Viability analysis of GICs treated with prazosin after sorting cells according to their expression of EGFR, and the neural stem cell markers CD15 and CD133. Prazosin inhibits cell viability regardless of CD133 or CD15 expression. *P=0.0286, n=4, two-sided Mann-Whitney U-test.


F . Imunocytochemical of NSCs GICs cultured in media favoring neuronal ( ß3 - tubulin ) , astroglial ( GFAP ) or oligodendroglial ( O4 ) differentiation ( Diff media ) . ß3 - tub : ß3 - tubulin .

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F. Imunocytochemical staining of NSCs and GICs cultured in media favoring neuronal (ß3-tubulin), astroglial (GFAP) or oligodendroglial (O4) differentiation (Diff media). ß3-tub: ß3-tubulin.


G . Viability of differentiated NSCs GICs treated with prazosin for 72 h . Diff : differentiation . * P = 0 . 0286 , n = 4 , two - sided Mann - Whitney U - .

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G. Viability analysis of differentiated NSCs and GICs treated with prazosin for 72 h. Diff: differentiation. *P=0.0286, n=4, two-sided Mann-Whitney U-test.


B , C . In vivo effect of prazosin treatment ( 1 . 5mg / Kg ) on glioblastoma growth . Tumors were initiated with GBM44 GICs ( B ) or GMB5 GICs ( C ) . Left panels : Bioluminescent in vivo of tumors in mice treated with prazosin ( PRZ ) or vehicle for 45 days . Middle panels : of bioluminescent signals . Fold change in total flux represents : total flux after treatment / total flux before treatment . * P = 0 . 0002 * P = 0 . 003 for GBM44 GBM5 respectively , n = 8 , two - sided Mann - Whitney U - . Right panels : Kaplan - Meyer survival curves of mice treated with prazosin ( PRZ ) or vehicle demonstrating a significant survival benefit of prazosin as compared to vehicle , log - rank Mantel - Cox . treatment period is shaded in gray .

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B, C. In vivo effect of prazosin treatment (1.5mg/Kg) on glioblastoma growth. Tumors were initiated with GBM44 GICs (B) or GMB5 GICs (C). Left panels: Bioluminescent in vivo images of tumors in mice treated with prazosin (PRZ) or vehicle for 45 days. Middle panels: Quantification of the bioluminescent signals. Fold change in total flux represents the ratio: total flux after treatment/total flux before treatment. *P=0.0002 and *P=0.003 for GBM44 and GBM5 respectively, n=8, two-sided Mann-Whitney U-test. Right panels: Kaplan-Meyer survival curves of mice treated with prazosin (PRZ) or vehicle demonstrating a significant survival benefit of prazosin as compared to vehicle, log-rank Mantel-Cox test. The treatment period is shaded in gray.


D . Example of hematoxilin / eosin of brain coronal sections from mice sacrificed after 45 days of treatment with prazosin ( PRZ ) or vehicle . i : infiltration . n : necrosis . Scale bar = 2 mm . GBM44 GICs .

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D. Example of hematoxilin/eosin staining of brain coronal sections from mice sacrificed after 45 days of treatment with prazosin (PRZ) or vehicle. i: tumor infiltration. n: tumor necrosis. Scale bar = 2 mm. GBM44 GICs.


E . Left panel : Representative flow cytometry plots depicting of CD133 + glioblastoma ( GFP + ) isolated from mice treated with prazosin ( PRZ ) or vehicle . Tumors were initiated with GBM44 GICs . Right panel : of flow cytometry analyses of CD133 expression by GFP + - isolated from xenografts of three prazosin - treated ( PRZ ) mice three vehicle - treated mice . * P = 0 . 0003 , n = 6 , two - sided Mann - Whitney U - .

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E. Left panel: Representative flow cytometry plots depicting the percentage of CD133+ glioblastoma cells (GFP+) isolated from mice treated with prazosin (PRZ) or vehicle. Tumors were initiated with GBM44 GICs. Right panel: Quantification of flow cytometry analyses of CD133 expression by GFP+-tumor cells isolated from xenografts of three prazosin-treated (PRZ) mice and three vehicle-treated mice. *P=0.0003, n=6, two-sided Mann-Whitney U-test.


F . Secondary grafting of ( GBM44 - GFP + ) isolated from vehicle or prazosin - treated mice bearing tumors . Mice injected with glioblastoma isolated from prazosin - treated mice developed tumors at a lower frequency ( 50 % versus 100 % ) . Moreover , mice injected with glioblastoma isolated from prazosin - treated mice presented a statistically significant survival benefit . Left Panel : Bioluminescent in vivo of secondary grafts . Middle panel : of bioluminescent signals . * P = 0 . 0004 , n = 8 , two - sided Mann - Whitney U - . Right panel : Kaplan - Meyer survival curves of mice bearing secondary graft from glioblastoma isolated from vehicle - prazosin - treated mice .

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F. Secondary grafting of tumor cells (GBM44-GFP+) isolated from vehicle or prazosin-treated mice bearing primary tumors. Mice injected with glioblastoma cells isolated from prazosin-treated mice developed tumors at a lower frequency (50% versus 100%). Moreover, mice injected with glioblastoma cells isolated from prazosin-treated mice presented a statistically significant survival benefit. Left Panel: Bioluminescent in vivo images of secondary grafts. Middle panel: Quantification of the bioluminescent signals. *P=0.0004, n=8, two-sided Mann-Whitney U-test. Right panel: Kaplan-Meyer survival curves of mice bearing secondary graft from glioblastoma cells isolated from vehicle- and prazosin-treated mice.


G . Inhibitory effect of low doses of prazosin ( 0 . 15 mg / Kg ) on glioblastoma growth in vivo . Left panel : Bioluminescent in vivo of tumors in mice treated with vehicle or prazosin ( PRZ ) for 45 days . Tumors were initiated with GBM44 GICs ( compare with panel B ) . Middle panel : of bioluminescent signals . * P = 0 . 0007 , n = 8 , two - sided Mann - Whitney U - . Right panel : Kaplan - Meyer survival curves of mice demonstrating a significant survival benefit of a treatment with low doses of prazosin , log - rank Mantel - Cox . treatment period is shaded in gray .

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G. Inhibitory effect of low doses of prazosin (0.15 mg/Kg) on glioblastoma growth in vivo. Left panel: Bioluminescent in vivo images of tumors in mice treated with vehicle or prazosin (PRZ) for 45 days. Tumors were initiated with GBM44 GICs (compare with panel B). Middle panel: Quantification of the bioluminescent signals. *P=0.0007, n=8, two-sided Mann-Whitney U-test. Right panel: Kaplan-Meyer survival curves of mice demonstrating a significant survival benefit of a treatment with low doses of prazosin, log-rank Mantel-Cox test. The treatment period is shaded in gray.


A . Viability of GL261 treated with prazosin in vitro . * P = 0 . 0286 , n = 4 , two - sided Mann - Whitney U - .

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A. Viability analysis of GL261 treated with prazosin in vitro. *P=0.0286, n=4, two-sided Mann-Whitney U-test.


C . Prazosin inhibits in vivo tumorgrowth in an immunocompetent model of glioblastoma . Left panel : Bioluminescent in vivo of tumors in C57 / Bl6mice treated with vehicle or prazosin ( PRZ ) for 14 days . Middle panel : of bioluminescent signals . * P = 0 . 0002 , n = 8 , two - sided Mann - Whitney U - . Right panel : Kaplan - Meyer survival curves of mice treated with prazosin ( PRZ ) or vehicle . log - rank Mantel - Cox . treatment period is shaded in gray .

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C. Prazosin inhibits in vivo tumorgrowth in an immunocompetent model of glioblastoma. Left panel: Bioluminescent in vivo images of tumors in C57/Bl6mice treated with vehicle or prazosin (PRZ) for 14 days.Middle panel: Quantification of the bioluminescent signals. *P=0.0002, n=8, two-sided Mann-Whitney U-test. Right panel: Kaplan-Meyer survival curves of mice treated with prazosin (PRZ) or vehicle. log-rank Mantel-Cox test. The treatment period is shaded in gray.


D . Hematoxilin / eosin of C57 / Bl6 mice brain coronal sections sacrificed after 14 days of treatment with prazosin . Scale bar = 2 mm .

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D. Hematoxilin/eosin staining of C57/Bl6 mice brain coronal sections sacrificed after 14 days of treatment with prazosin. Scale bar = 2 mm.


E . imaging of GL261 intra - striatal grafts at 1 2 h after an intra - peritoneal injection of BODIPY FL prazosin , green - derivative of prazosin . Scale bar = 2 mm .

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E. Fluorescence imaging of GL261 intra-striatal grafts at 1 and 2 h after an intra-peritoneal injection of BODIPY FL prazosin, the green-fluorescent derivative of prazosin. Scale bar = 2 mm.


A . Immunoblotting for pro - caspase - 3 ( pro - CASP3 ) active caspase - 3 ( CASP3 ) in GICs treated with prazosin ( PRZ ) or vehicle ( V ) demonstrating that prazosin activates caspase - 3 . ZVAD , a caspase inhibitor , prevents prazosin - induced caspase - 3 activation . kDa : kilodaltons .

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A. Immunoblotting for pro-caspase-3 (pro-CASP3) and active caspase-3 (CASP3) in GICs treated with prazosin (PRZ) or vehicle (V) demonstrating that prazosin activates caspase-3. ZVAD, a caspase inhibitor, prevents prazosin-induced caspase-3 activation. kDa: kilodaltons.


B . Immunoblotting for pro - caspase - 9 ( pro - CASP9 ) active caspase - 9 ( CASP9 ) in GICs treated with prazosin ( PRZ ) or vehicle ( V ) demonstrating that prazosin does not activate caspase - 9 .

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B. Immunoblotting for pro-caspase-9 (pro-CASP9) and active caspase-9 (CASP9) in GICs treated with prazosin (PRZ) or vehicle (V) demonstrating that prazosin does not activate caspase-9.


C . Viability of GICs treated with prazosin for 24h in presence or absence of ZVAD , a caspase inhibitor . ZVAD counteracts prazosin - induced GIC . * P = 0 . 0286 , n = 4 , two - sided Mann - Whitney U - .

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C. Viability analysis of GICs treated with prazosin for 24h in the presence or absence of ZVAD, a caspase inhibitor. ZVAD counteracts prazosin-induced GIC death. *P=0.0286, n=4, two-sided Mann-Whitney U-test.


D . Prazosin induces glioblastoma apoptosis in vivo . GFP + GFP - were analyzed from tumors in vehicle or prazosin - treated mice . Annexin V / DAPI was used to identify apoptotic by FACS . Tumors were initiated by GBM44 GICs implantation .

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D. Prazosin induces glioblastoma cell apoptosis in vivo. GFP+ and GFP- cells were analyzed from tumors in vehicle or prazosin-treated mice. Annexin V / DAPI staining was used to identify apoptotic cells by FACS. Tumors were initiated by GBM44 GICs implantation.


E . Dose - response curve of prazosin on GIC survival ( 24h treatment ) . EC50 was determined with curve fit using nonlinear regression .

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E. Dose-response curve of prazosin on GIC survival (24h treatment). EC50 was determined with curve fit using nonlinear regression.


F . Viability of GICs treated with prazosin for 24h in presence or absence of cirazoline , a subtype agonist of α - ARs . Cirazoline did not alter GIC survival counteracted only poorly prazosin - induced GIC . * P < 0 . 05 , n = 4 , two - sided Mann - Whitney U - .

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F. Viability analysis of GICs treated with prazosin for 24h in the presence or absence of cirazoline, a subtype agonist of the α-ARs. Cirazoline did not alter GIC survival and counteracted only poorly prazosin-induced GIC death. *P<0.05, n=4, two-sided Mann-Whitney U-test.


G . binding assays demonstrating absence of prazosin binding sites in GIC preparations . Positive control shows that prazosin binds to preparations of yeast expressing 1 - AR . PRZ : prazosin . CRZ : cirazoline .

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G. Membrane binding assays demonstrating the absence of prazosin binding sites in GIC membrane preparations. Positive control shows that prazosin binds to membrane preparations of yeast expressing 1-AR. PRZ: prazosin. CRZ: cirazoline.


H . Immunoblotting for phosphorylated ERK1 / 2 ( P - ERK1 / 2 ) total ERK1 / 2 following prazosin treatment for 30 min . Prazosin induces ERK1 / 2 phosphorylation in GICs .

H. Immunoblotting for phosphorylated ERK1/2 (P-ERK1/2) and total ERK1/2 following prazosin treatment for 30 min. Prazosin induces ERK1/2 phosphorylation in GICs.


I . SRE - luciferase reporter activity of GICs treated with prazosin in absence or presence of U0126 ( 10 μM ) , an inhibitor of ERK - activating kinase MEK . U0126 prevented prazosin - induced SRE - luciferase activation in GICs .

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I. SRE-luciferase reporter activity analysis of GICs treated with prazosin in absence or presence of U0126 (10 μM), an inhibitor of the ERK-activating kinase MEK. U0126 prevented prazosin-induced SRE-luciferase activation in GICs.


J . Viability of GICs treated with prazosin for 24 h in presence or absence of U0126 ( 10 μM ) , an inhibitor of ERK - activating kinase MEK . U0126 did not counteract prazosin - induced GIC . Blockade of prazosin - induced ERK1 / 2 phosphorylation by U0126 was confirmed by immunoblotting ( insert ) . PRZ : prazosin .

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J. Viability analysis of GICs treated with prazosin for 24 h in the presence or absence of U0126 (10 μM), an inhibitor of the ERK-activating kinase MEK. U0126 did not counteract prazosin-induced GIC death. Blockade of prazosin-induced ERK1/2 phosphorylation by U0126 was confirmed by immunoblotting (insert). PRZ: prazosin.


A . PKCδ expression in two - derived GICs NSCs by immunoblotting . GICs express higher of PKCδ than NSCs . kDa : kilodaltons .

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A. PKCδ expression analysis in two patient-derived GICs and NSCs by immunoblotting. GICs express higher levels of PKCδ than NSCs. kDa: kilodaltons.


B . PKCδ by immunostaining ( green ) . Nuclei were stained with DAPI ( blue ) . Enhanced PKCδ punctate expression in prazosin - treated ( PRZ , 5µM ) GICs , as compared to vehicle - treated GICs . PRZ : prazosin . Scale bar : 10 µm .

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B. PKCδ by immunostaining (green). Nuclei were stained with DAPI (blue). Enhanced PKCδ punctate nuclear expression in prazosin-treated (PRZ, 5µM) GICs, as compared to vehicle-treated GICs. PRZ: prazosin. Scale bar: 10 µm.


C . Total PKCδ ( PKCδ 78kDa ) PKCδ ( PKCδ 41kDa ) expression in GICs by immunoblotting . Prazosin ( PRZ ) induces PKCδ cleavage into a 41kDa active fragment . 2 µM rottlerin ( Rott ) , a PKCδ inhibitor , inhibits prazosin - induced PKCδ cleavage . V : vehicle . kDa : kilodaltons .

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C. Total PKCδ (PKCδ 78kDa) and cleaved PKCδ (PKCδ 41kDa) expression analysis in GICs by immunoblotting. Prazosin (PRZ) induces PKCδ cleavage into a 41kDa active fragment. 2 µM rottlerin (Rott), a PKCδ inhibitor, inhibits prazosin-induced PKCδ cleavage. V: vehicle. kDa: kilodaltons.


D . Pro - caspase - 3 ( Pro - CASP3 ) activated caspase - 3 ( CASP3 ) expression in GICs by immunoblotting . 2 µM rottlerin ( Rott ) inhibits prazosin ( PRZ ) - induced caspase - 3 activation . V : vehicle . kDa : kilodaltons .

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D. Pro-caspase-3 (Pro-CASP3) and activated caspase-3 (CASP3) expression analysis in GICs by immunoblotting. 2 µM rottlerin (Rott) inhibits prazosin (PRZ)-induced caspase-3 activation. V: vehicle. kDa: kilodaltons.


E . Inhibition of PKCδ by rottlerin prevents , in a - dependent manner , prazosin - induced GIC . Viability of GICs treated with prazosin for 72 h in presence or absence of rottlerin . * P = 0 . 0286 , n = 4 , two - sided Mann - Whitney U - .

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E. Inhibition of PKCδ by rottlerin prevents, in a concentration-dependent manner, prazosin-induced GIC death. Viability analysis of GICs treated with prazosin for 72 h in the presence or absence of rottlerin. *P=0.0286, n=4, two-sided Mann-Whitney U-test.


F . Inhibition of PKCδ by specific anti - PKCδ RACK peptide δV1 . 1 ( 10 μM ) counteracts prazosin - induced GIC . Viability of GICs treated with prazosin for 72 h in presence or absence of δV1 . 1 . * P < 0 . 005 for Prazosin 1 5 µM P < 0 . 001 for Prazosin 10 µM by two - unpaired Student ' s t - , n = 4 .

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F. Inhibition of PKCδ by the specific anti-PKCδ RACK peptide δV1.1 (10 μM) counteracts prazosin-induced GIC death. Viability analysis of GICs treated with prazosin for 72 h in the presence or absence of δV1.1. *P < 0.005 for Prazosin 1 and 5 µM and P<0.001 for Prazosin 10 µM by two-tailed unpaired Student's t-test, n=4.


G . Decreased PKCδ expression using shRNA counteracts prazosin - induced GIC . Viability of GICs transduced with scrambled or PKCδ shRNA treated with prazosin for 72 h . * P < 0 . 005 by two - unpaired Student ' s t - , n = 4 .

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G. Decreased PKCδ expression using shRNA counteracts prazosin-induced GIC death. Viability analysis of GICs transduced with scrambled or PKCδ shRNA and treated with prazosin for 72 h. *P < 0.005 by two-tailed unpaired Student's t-test, n=4.


H . Prazosin inhibits AKT phosphorylation in GICs . LY294002 ( LY , 30 µM ) , an inhibitor of PI3K / AKT pathway , was used as positive control . Terazosin , which does not affect GIC viability , does not alter AKT phosphorylation ( see corresponding viability counting in Fig EV4C ) . Phosphorylated AKT ( P - AKT ) total AKT ( AKT ) expression in GICs by immunoblotting . V : vehicle . kDa : kilodaltons .

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

H. Prazosin inhibits AKT phosphorylation in GICs. LY294002 (LY, 30 µM), an inhibitor of the PI3K/AKT pathway, was used as positive control. Terazosin, which does not affect GIC viability, does not alter AKT phosphorylation (see the corresponding cell viability counting in Fig EV4C). Phosphorylated AKT (P-AKT) and total AKT (AKT) expression analysis in GICs by immunoblotting. V: vehicle. kDa: kilodaltons.


I . Prazosin ( 10 µM ) does not inhibit AKT phosphorylation in NSCs . by immunoblotting . V : vehicle . kDa : kilodaltons .

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

I. Prazosin (10 µM) does not inhibit AKT phosphorylation in NSCs. Analysis by immunoblotting. V: vehicle. kDa: kilodaltons.


J . β - catenin expression , a downstream target of AKT , is decreased in prazosin - treated GICs , as opposed to NSCs . by immunoblotting . V : vehicle . kDa : kilodaltons .

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

J. β-catenin expression, a downstream target of AKT, is decreased in prazosin-treated GICs, as opposed to NSCs. Analysis by immunoblotting. V: vehicle. kDa: kilodaltons.


K . L . Inhibition of PKCδ using rottlerin ( Rott , 2 µM ) or δV1 . 1 ( 10 µM ) counteracts prazosin inhibition of AKT phosphorylation . by immunoblotting . V : vehicle . kDa : kilodaltons .

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

K. L. Inhibition of PKCδ using rottlerin (Rott, 2 µM) or δV1.1 (10 µM) counteracts prazosin inhibition of AKT phosphorylation. Analysis by immunoblotting. V: vehicle. kDa: kilodaltons.


B . Bioluminescent in vivo of tumors in mice grafted with GICs expressing either scrambled or PKCδ shRNA . mice were treated with prazosin ( PRZ ) for 45 days . C . of bioluminescent signals showing that decreased expression of PKC counteracts prazosin inhibition of growth . Fold change in total flux represents : total flux after treatment / total flux prior treatment . * P = 0 . 0007 , n = 8 , two - sided Mann - Whitney U - .

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 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

B. Bioluminescent in vivo images of tumors in mice grafted with GICs expressing either scrambled or PKCδ shRNA. All mice were treated with prazosin (PRZ) for 45 days.C. Quantification of the bioluminescent signals showing that decreased expression of PKC counteracts prazosin inhibition of tumor growth. Fold change in total flux represents the ratio: total flux after treatment/total flux prior treatment. *P=0.0007, n=8, two-sided Mann-Whitney U-test.


of four - week - old soil - grown of Col - 0 , snc1 , two independent transgenic lines of SNIPER1 OE into snc1 background . OE stands for overexpression of SNIPER1 . Scale bar = 1 cm . SNIPER1 gene expression in indicated as determined by RT - PCR . Error bars represent mean ± SD ( n = 3 ) . Two independent experiments were carried out with similar results . of H . a . Noco2 sporulation in indicated genotypes 7 days post inoculation ( dpi ) with 105 spores per ml water . Error bars represent mean ± SD ( n = 4 ) . Three independent experiments were carried out with similar results . Growth of P . s . m . ES4326 on four - week - old leaves of indicated genotypes at 0 3 dpi with inoculum of OD600 = 0 . 001 . Error bars represent mean ± SD ( n = 4 for day 0 ; n = 5 for day 3 ) . Three independent experiments were carried out with similar results .

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

Morphology of four-week-old soil-grown plants of Col-0, snc1, and two independent transgenic lines of SNIPER1 OE into snc1 background. OE stands for overexpression of SNIPER1. Scale bar = 1 cm. SNIPER1 gene expression in the indicated plants as determined by RT-PCR. Error bars represent mean ± SD (n = 3). Two independent experiments were carried out with similar results. Quantification of H.a. Noco2 sporulation in the indicated genotypes 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results. Growth of P.s.m. ES4326 on four-week-old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0.001. Error bars represent mean ± SD (n=4 for day 0; n=5 for day 3). Three independent experiments were carried out with similar results.


of four - week - old soil - grown of two independent transgenic lines of SNIPER1 OE in Col - 0 background . Scale bar = 1 cm . No difference in growth development was observed . SNIPER1 gene expression in indicated as determined by RT - PCR . Error bars represent mean ± SD ( n = 3 ) . Two independent experiments were carried out with similar results . of H . a . Noco2 sporulation in indicated genotypes 7 days post inoculation ( dpi ) with 105 spores per ml water . Error bars represent mean ± SD ( n = 4 ) . Three independent experiments were carried out with similar results . Growth of P . s . t . DC3000 on four - week - old leaves of indicated genotypes at 0 3 dpi with inoculum of OD600 = 0 . 001 . Error bars represent mean ± SD ( n = 4 for day 0 ; n = 5 for day 3 ) . Three independent experiments were carried out with similar results .

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 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

Morphology of four-week-old soil-grown plants of two independent transgenic lines of SNIPER1 OE in Col-0 background. Scale bar = 1 cm. No difference in growth and development was observed. SNIPER1 gene expression in the indicated plants as determined by RT-PCR. Error bars represent mean ± SD (n = 3). Two independent experiments were carried out with similar results. Quantification of H.a. Noco2 sporulation in the indicated genotypes 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results. Growth of P.s.t. DC3000 on four-week-old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0.001. Error bars represent mean ± SD (n=4 for day 0; n=5 for day 3). Three independent experiments were carried out with similar results.


of four - week - old soil - grown of Col - 0 , chs1 - 2 ( A ) , chs2 - 1 ( B ) two independent transgenic lines of SNIPER1 OE into chs1 - 2 ( A ) chs2 - 1 ( B ) backgrounds . were grown at 16 ° C under long day conditions . Scale bar = 1 cm .

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

Morphology of four-week-old soil-grown plants of Col-0, chs1-2 (A), chs2-1 (B) and two independent transgenic lines of SNIPER1 OE into chs1-2 (A) and chs2-1 (B) backgrounds. Plants were grown at 16 °C under long day conditions. Scale bar = 1 cm.


of four - week - old soil - grown of Col - 0 , chs3 - 2D ( C ) , snc2 - 1D eds5 npr1 ( D ) two independent transgenic lines of SNIPER1 OE into chs3 - 2D ( C ) snc2 - 1D eds5 npr1 ( D ) backgrounds . Scale bar = 1 cm .

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

Morphology of four-week-old soil-grown plants of Col-0, chs3-2D (C), snc2-1D eds5 npr1 (D) and two independent transgenic lines of SNIPER1 OE into chs3-2D (C) and snc2-1D eds5 npr1 (D) backgrounds. Scale bar = 1 cm.


of four - week - old soil - grown of Col - 0 , mekk1 - 5 ndr1 , two independent transgenic lines of SNIPER1 OE into mekk1 - 5 ndr1 background . Scale bar = 1 cm . mekk1 ndr1 double mutant was used instead of mekk1 - 5 single mutant is to overcome sterility . SNIPER1 gene expression in indicated as determined by RT - PCR . Error bars represent mean ± SD ( n = 3 ) . Two independent experiments were carried out with similar results . of H . a . Noco2 sporulation in indicated genotypes 7 days post inoculation ( dpi ) with 105 spores per ml water . Error bars represent mean ± SD ( n = 4 ) . Three independent experiments were carried out with similar results . Growth of P . s . m . ES4326 on four - week - old leaves of indicated genotypes at 0 3 dpi with inoculum of OD600 = 0 . 001 . mn stands for mekk1 - 5 ndr1 double mutant . Error bars represent mean ± SD ( n = 4 ) . Three independent experiments were carried out with similar results .

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

Morphology of four-week-old soil-grown plants of Col-0, mekk1-5 ndr1, and two independent transgenic lines of SNIPER1 OE into mekk1-5 ndr1 background. Scale bar = 1 cm. The mekk1 ndr1 double mutant was used instead of the mekk1-5 single mutant is to overcome the sterility. SNIPER1 gene expression in the indicated plants as determined by RT-PCR. Error bars represent mean ± SD (n = 3). Two independent experiments were carried out with similar results. Quantification of H.a. Noco2 sporulation in the indicated genotypes 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results. Growth of P.s.m. ES4326 on four-week-old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0.001. mn stands for mekk1-5 ndr1 double mutant. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results.


In vitro ubiquitination using E . coli - expressed SNIPER1 - myc ( E3 ) , AtUBC8 ( E2 ) , AtUBA1 ( E1 ) / or HIS - FLAG - Ubiquitin . molecular mass markers are indicated on left ( kDa ) . Asterisk indicates SNIPER1 - myc . Arrow points to His - FLAG - Ub straight lines on right indicate self - ubiquitinated SNIPER1 .

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

In vitro ubiquitination assay using E. coli-expressed SNIPER1-myc (E3), AtUBC8 (E2), AtUBA1 (E1) and/or HIS-FLAG-Ubiquitin. The molecular mass markers are indicated on the left (kDa). Asterisk indicates SNIPER1-myc. Arrow points to His-FLAG-Ub and straight lines on the right indicate self-ubiquitinated SNIPER1.


Immunoblot of SNC1 ( A - B ) , SUMM2 - 3HA ( C ) , RPS4HA ( D ) , RPP4myc ( E ) , RPS2HA ( F ) RPM1myc ( G ) in indicated genotypes . For panels , equal loading is shown by Ponceau S of a non - specific band . numbers below represent normalized between of band Ponceau S band ± SD ( n = 3 ) . Molecular mass marker in kilo Daltons is indicated on left . Three independent experiments were carried out with similar results .

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

Immunoblot analysis of SNC1 (A-B), SUMM2-3HA (C), RPS4HA (D), RPP4myc (E), RPS2HA (F) and RPM1myc (G) protein levels in the indicated genotypes. For all panels, equal loading is shown by Ponceau S staining of a non-specific band. The numbers below represent the normalized ratio between the intensity of the protein band and the Ponceau S band ± SD (n=3). Molecular mass marker in kilo Daltons is indicated on the left. Three independent experiments were carried out with similar results.


Growth of P . s . t . avrRps4 ( A ) , avrRpt2 ( B ) avrRpm1 ( C ) on four - week - old leaves of indicated genotypes at 3 dpi with inoculum of OD600 = 0 . 0001 . Error bars represent mean ± SD ( n = 5 ) . Three independent experiments were carried out with similar results . Appendix Figure S4 shows growth of P . s . t . avrRps4 , avrRpt2 avrRpm1 at 0 dpi . of H . a . Emwa1 sporulation in indicated genotypes 7 days post inoculation ( dpi ) with 105 spores per ml water . Error bars represent mean ± SD ( n = 4 ) . Three independent experiments were carried out with similar results .

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

Growth of P.s.t. avrRps4 (A), avrRpt2 (B) and avrRpm1 (C) on four-week-old leaves of the indicated genotypes at 3 dpi with bacterial inoculum of OD600 = 0.0001. Error bars represent mean ± SD (n=5). Three independent experiments were carried out with similar results. Appendix Figure S4 shows the growth of P.s.t. avrRps4, avrRpt2 and avrRpm1 at 0 dpi. Quantification of H.a. Emwa1 sporulation in the indicated genotypes 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results.


of four - week - old soil - grown of Col - 0 two independent transgenic lines of 35S - SNIPER1H129Y . Scale bar = 1 cm .

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

Morphology of four-week-old soil-grown plants of Col-0 and two independent transgenic lines of 35S-SNIPER1H129Y. Scale bar = 1 cm.


PR1 PR2 gene expression in indicated as determined by RT - PCR . Error bars represent mean ± SD ( n = 3 ) . Two independent experiments were carried out with similar results .

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

PR1 and PR2 gene expression in the indicated plants as determined by RT-PCR. Error bars represent mean ± SD (n = 3). Two independent experiments were carried out with similar results.


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

Morphology of four-week-old soil-grown plants of Col-0, sniper1-1, sniper2-1, eds1-2, SNIPER1H129Y, SNIPER1H129Y eds1-2, sniper1-1 sniper2-1 eds1-2 and sniper1-c1 sniper2-1 eds1-2. sniper1-1 is an exonic T-DNA insertional mutant. sniper1-c1 is a SNIPER1 knockout mutant containing 807bp deletion that was generated by CRISPR-Cas9 system. Scale bar = 1 cm.


of H . a . Noco2 sporulation in indicated 7 days post inoculation ( dpi ) with 105 spores per ml water . Error bars represent mean ± SD ( n = 4 ) . Two independent experiments were carried out with similar results .

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

Quantification of H.a. Noco2 sporulation in the indicated plants 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Two independent experiments were carried out with similar results.


Immunoblot of RPS4HA ( E ) , SNC1 ( F ) , RPS2HA ( G ) , RPM1myc ( H ) in SNIPER1H129Y background . Equal loading is shown by Ponceau S of a non - specific band . numbers below represent normalized between of band Ponceau S band ± SD ( n = 3 ) . Molecular mass marker in kilo Daltons is indicated on left . Three independent experiments were carried out with similar results .

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

Immunoblot of RPS4HA (E), SNC1 (F), RPS2HA (G), and RPM1myc (H) in SNIPER1H129Y background. Equal loading is shown by Ponceau S staining of a non-specific band. The numbers below represent the normalized ratio between the intensity of the protein band and the Ponceau S band ± SD (n=3). Molecular mass marker in kilo Daltons is indicated on the left. Three independent experiments were carried out with similar results.


Immunoprecipitation biotinylation of SUMM2NB - FLAG ( A ) , RPP4NB - FLAG ( B ) , or CHS1NB - FLAG ( C ) by SNIPER1 H129Y - HATurboID in N . benthamiana . Two biological repeats were carried out with similar results .

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

Immunoprecipitation and biotinylation of SUMM2NB-FLAG (A), RPP4NB-FLAG (B), or CHS1NB-FLAG (C) by SNIPER1 H129Y-HATurboID in N. benthamiana. Two biological repeats were carried out with similar results.


Interaction of SNIPER1 H129Y - CLuc SUMM2NB - NLuc ( D ) , RPP4NB - NLuc ( E ) or CHS1NB - NLuc ( F ) as tested by split - luciferase complementary in N . benthamiana . Three biological repeats with four technical repeats each were carried out with similar results .

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

Interaction of SNIPER1 H129Y-CLuc and SUMM2NB-NLuc (D), RPP4NB-NLuc (E) or CHS1NB-NLuc (F) as tested by split-luciferase complementary assay in N. benthamiana. Three biological repeats with four technical repeats each were carried out with similar results.


Ubiquitination of NB domains from SUMM2 ( A ) , RPP4 ( B ) CHS1 ( C ) . Immunoblot of lysates form E . coil strains expressing AtUBA1 - S , MBP - NB - HA , AtUBC8 - S , HIS - FLAG - UBQ10 SNIPER1 - myc , or strains without one of these components . Two biological repeats were carried out with similar results .

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

Ubiquitination of the NB domains from SUMM2 (A), RPP4 (B) and CHS1 (C). Immunoblot of bacterial lysates form E. coil strains expressing AtUBA1-S, MBP-NB-HA, AtUBC8-S, HIS-FLAG-UBQ10 and SNIPER1-myc, or strains without one of these components. Two biological repeats were carried out with similar results.


( G ) Reduced NMDA / AMPA at SC - CA1 synapses of PtenΔC / ΔC mice ( P17 - 21 ) . NMDAR component was obtained 60 ms after stimulation . ( , n = 11 from 8 mice for WT 8 ( 8 ) for ΔC ; decay , n = 12 from 8 mice for WT 11 ( 8 ) for ΔC , * * P < 0 . 01 , ns , not significant , Mann - Whitney U ) . error bars represent . ( H ) Reduced amplitude but not frequency of NMDA - mEPSCs at PtenΔC / ΔC CA1 pyramidal neurons . ( n = 17 from 4 mice for WT 18 ( 4 ) for ΔC , * * P < 0 . 01 , ns , not significant , Mann - Whitney U , Student ' s t - ) . error bars represent . ( I ) Reduced NMDA / AMPA at synapses in layer II / III pyramidal neurons in prelimbic region of mPFC of PtenΔC / ΔC mice ( P20 - 21 ) . NMDAR component was obtained 60 ms after stimulation . ( n = 9 from 3 mice for WT 10 ( 3 ) for ΔC , * P < 0 . 05 , * * P < 0 . 01 , Student ' s t - , Mann - Whitney U ) . error bars represent . Data information : ( C - F ) Results from last 10 - minute recordings are shown in scatter plots .

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

(G) Reduced NMDA/AMPA ratio at SC-CA1 synapses of PtenΔC/ΔC mice (P17-21). The NMDAR component was obtained 60 ms after stimulation. (ratio, n = 11 cells from 8 mice for WT and 8 (8) for ΔC; decay, n = 12 cells from 8 mice for WT and 11 (8) for ΔC, **P < 0.01, ns, not significant, Mann-Whitney U test). The error bars represent SEM. (H) Reduced amplitude but not frequency of NMDA -mEPSCs at PtenΔC/ΔC CA1 pyramidal neurons. (n = 17 cells from 4 mice for WT and 18 (4) for ΔC, **P < 0.01, ns, not significant, Mann-Whitney U test, Student's t-test). The error bars represent SEM. (I) Reduced NMDA/AMPA ratio at synapses in layer II/III pyramidal neurons in the prelimbic region of the mPFC of PtenΔC/ΔC mice (P20-21). The NMDAR component was obtained 60 ms after stimulation. (n = 9 cells from 3 mice for WT and 10 (3) for ΔC, *P < 0.05, **P < 0.01, Student's t-test, Mann-Whitney U test). The error bars represent SEM. Data information: (C-F) Results from the last 10-minute recordings are shown in scatter plots.


A ) A volcano plot showing DEGs ( adjusted p value < 0 . 1 ) derived from - Seq results for PtenΔC / ΔC WT mice ( P21 ) . For calculation of adjusted p value , we have employed R package DESeq2 where P ‐ values obtained by Wald are corrected for multiple testing using Benjamini Hochberg method . ( n = 3 mice for WT ΔC ) . See also Dataset EV1 for full - Seq results .

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

A) A volcano plot showing DEGs (adjusted p value < 0.1) derived from RNA-Seq results for PtenΔC/ΔC and WT mice (P21). For the calculation of adjusted p value, we have employed the R package DESeq2 where the P‐values obtained by the Wald test are corrected for multiple testing using the Benjamini and Hochberg method. (n = 3 mice for WT and ΔC). See also Dataset EV1 for the full RNA-Seq results.


C ) Immunoblot validation of increase in SLC6A20 in hippocampal lysates of PtenΔC / ΔC mice ( P21 ) . We denoted antibody as ' SLC6A20 ' here , not ' SLC6A20A ' , because this antibody was generated using a synthetic peptide ( YNEPSNNCQKHAI ) that is commonly present in SLC6A20A SLC6A20B , being a pan - SLC6A20 antibody ( ThermoFisher ) . ( n = 3 mice for WT ∆ C , * P < 0 . 05 , Student ' s t - ) . error bars represent .

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

C) Immunoblot validation of the increase in SLC6A20 protein levels in hippocampal lysates of PtenΔC/ΔC mice (P21). We denoted the antibody as 'SLC6A20' here, not 'SLC6A20A', because this antibody was generated using a synthetic peptide sequence (YNEPSNNCQKHAI) that is commonly present in SLC6A20A and SLC6A20B, being a pan-SLC6A20 antibody (ThermoFisher). (n = 3 mice for WT and ∆C, *P < 0.05, Student's t-test). The error bars represent SEM.


( D , E ) Increased phosphorylation of β - catenin at Ser - 675 but not at Ser - 552 in nucleus - enriched P1 fraction but not in whole - brain lysates of PtenΔC / ΔC mice ( 3 months ) . Note that total of β - catenin were not changed in P1 fraction or whole - brain lysates . ( n = 4 mice for WT - WB / P1 ΔC - WB / P1 , * P < 0 . 05 , ns , not significant , Student ' s t - ) . error bars represent .

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 B-CONTROLLED_VAR 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

(D,E) Increased phosphorylation of β-catenin at Ser-675 but not at Ser-552 in the nucleus-enriched P1 fraction but not in whole-brain lysates of PtenΔC/ΔC mice (3 months). Note that total levels of β-catenin were not changed in the P1 fraction or whole-brain lysates. (n = 4 mice for WT-WB/P1 and ΔC-WB/P1, *P < 0.05, ns, not significant, Student's t-test). The error bars represent SEM.


( B , C ) Decreased extracellular of proline glycine in brains of PtenΔC / ΔC mice ( 2 - 4 month ) , as determined by microdialysis in hippocampus ( see Materials Methods for details ) . ( proline , n = 13 mice for WT 8 for ΔC , * * P < 0 . 01 , Student ' s t - ; glycine , n = 9 for WT 5 for ∆ C , * * P < 0 . 01 , Student ' s t - , Mann - Whitney U ) . error bars represent .

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

(B,C) Decreased extracellular levels of proline and glycine in the brains of PtenΔC/ΔC mice (2-4 month), as determined by microdialysis in the hippocampus (see Materials and Methods for details). (proline, n = 13 mice for WT and 8 for ΔC, **P < 0.01, Student's t-test; glycine, n = 9 for WT and 5 for ∆C, **P < 0.01, Student's t-test, Mann-Whitney U test). The error bars represent SEM.


( B ) Normalization of NMDAR - dependent HFS - LTP at synapses of PtenΔC / ΔC mice ( 4 - 5 ) by DCS treatment ( 20 μM ) , as shown by fEPSP slopes . ( n = 15 slices from 9 mice for WT - V / vehicle , 11 ( 5 ) for WT - D / DCS , 16 ( 10 ) for ΔC - V , 13 ( 7 ) for ΔC - D , * * P < 0 . 01 , ns , not significant , two - way ANOVA with Tukey ' s ) . grey traces represent baseline fEPSP prior to LTP induction . error bars represent .

O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Normalization of NMDAR-dependent HFS-LTP at synapses of PtenΔC/ΔC mice (4-5 weeks) by DCS treatment (20 μM), as shown by fEPSP slopes. (n = 15 slices from 9 mice for WT-V/vehicle, 11 (5) for WT-D/DCS, 16 (10) for ΔC-V, 13 (7) for ΔC-D, **P < 0.01, ns, not significant, two-way ANOVA with Tukey's test). The grey traces represent the baseline fEPSP prior to LTP induction. The error bars represent SEM.


( F ) Normalization of NMDAR - dependent HFS - LTP at synapses of PtenΔC / ΔC mice ( 4 - 5 ) by sarcosine treatment ( 750 µM ) , as shown by fEPSP slopes . Note that data for WT - V ΔC - V are identical to those shown in Fig . 4B because whole experiments were performed together ; we generated independent figures for DCS sarcosine results for clear presentation of data . ( n = 15 slices from 9 mice for WT - V / vehicle , 9 ( 4 ) for WT - Sarc / Sarcosine , 16 ( 10 ) for ΔC - V / Vehicle , 18 ( 4 ) for ΔC - S , * P < 0 . 05 , * * * P < 0 . 001 , ns , not significant , two - way ANOVA with Tukey ' s ) . grey traces represent baseline fEPSP prior to LTP induction . error bars represent .

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

(F) Normalization of NMDAR-dependent HFS-LTP at synapses of PtenΔC/ΔC mice (4-5 weeks) by sarcosine treatment (750 µM), as shown by fEPSP slopes. Note that the data for WT-V and ΔC-V are identical to those shown in Fig. 4B because the whole experiments were performed together; we generated independent figures for DCS and sarcosine results for the clear presentation of the data. (n = 15 slices from 9 mice for WT-V/vehicle, 9 (4) for WT-Sarc/Sarcosine, 16 (10) for ΔC-V/Vehicle, 18 (4) for ΔC-S, *P < 0.05, ***P < 0.001, ns, not significant, two-way ANOVA with Tukey's test). The grey traces represent the baseline fEPSP prior to LTP induction. The error bars represent SEM.


( D ) PtenΔC / ΔC mice ( 2 - 4 months ) treated with Slc6a20a - ASO display partially normalized of whole - brain glycine , as shown by lack of difference between saline - treated WT ASO - treated PtenΔC / ΔC mice . Note that glycine observed here are ~ 4 times lower than those measured in Fig . 3A , which could be attributable to different mouse ages ( P21 in Fig . 3A vs . 2 - 4 months in Fig . 5D ) , absence presence of ASO injection , or lot - to - lot variation of ELISA kits . ( n = 8 mice for WT - saline , 5 for WT - ASO , 7 for ΔC - saline , 8 for ΔC - ASO , * * P < 0 . 01 , ns , not significant , two - way ANOVA with Tukey ' s ) . error bars represent .

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

(D) PtenΔC/ΔC mice (2-4 months) treated with Slc6a20a-ASO display partially normalized levels of whole-brain glycine, as shown by the lack of difference between saline-treated WT and ASO-treated PtenΔC/ΔC mice. Note that the glycine levels observed here are ~4 times lower than those measured in Fig. 3A, which could be attributable to different mouse ages (P21 in Fig. 3A vs. 2-4 months in Fig. 5D), absence and presence of ASO injection, or lot-to-lot variation of the ELISA kits. (n = 8 mice for WT-saline, 5 for WT-ASO, 7 for ΔC-saline, and 8 for ΔC-ASO, **P < 0.01, ns, not significant, two-way ANOVA with Tukey's test). The error bars represent SEM.


( E ) PtenΔC / ΔC mice ( P20 - 37 ) treated with Slc6a20a - ASO display an NMDA / AMPA at hippocampal SC - CA1 synapses that is comparable to that in ASO - treated WT mice . ( n = 11 neurons from 5 mice for WT - ASO , 10 ( 4 ) for ΔC - ASO , ns , not significant , Mann - Whitney ' s U ) . error bars represent .

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(E) PtenΔC/ΔC mice (P20-37) treated with Slc6a20a-ASO display an NMDA/AMPA ratio at hippocampal SC-CA1 synapses that is comparable to that in ASO-treated WT mice. (n = 11 neurons from 5 mice for WT-ASO, 10 (4) for ΔC-ASO, ns, not significant, Mann-Whitney's U test). The error bars represent SEM.


( G ) PtenΔC / ΔC mice ( 2 - 4 months ) treated with Slc6a20a - ASO display normal of climbing in Laboras . n = 6 mice for WT - saline , 7 for WT - ASO , 7 for ΔC - saline , 8 for ΔC - ASO , * P < 0 . 05 , * * P < 0 . 01 , ns , not significant , two - way ANOVA with Bonferroni ' s . error bars represent .

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

(G) PtenΔC/ΔC mice (2-4 months) treated with Slc6a20a-ASO display normal levels of climbing in the Laboras test. n = 6 mice for WT-saline, 7 for WT-ASO, 7 for ΔC-saline, and 8 for ΔC-ASO, *P < 0.05, **P <0.01, ns, not significant, two-way ANOVA with Bonferroni's test. The error bars represent SEM.


( D ) Increased extracellular of glycine proline in brain of Slc6a20a + / - Slc6a20a - / - mice , as shown by microdialysis analyses . Note that glycine are increased in both Slc6a20a + / - Slc6a20a - / - mice , whereas proline are increased only in Slc6a20a - / - mice . ( glycine , n = 19 mice for WT , 11 for HT , 8 for KO , * * P < 0 . 01 , Mann - Whitney U ; proline , n = 15 mice for WT , 11 for HT , 14 for KO , * * * P < 0 . 001 , ns , not significant , Mann - Whitney U ) . error bars represent .

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

(D) Increased extracellular levels of glycine and proline in the brain of Slc6a20a+/- and Slc6a20a-/- mice, as shown by microdialysis analyses. Note that glycine levels are increased in both Slc6a20a+/- and Slc6a20a-/- mice, whereas proline levels are increased only in Slc6a20a-/- mice. (glycine, n = 19 mice for WT, 11 for HT, 8 for KO, **P < 0.01, Mann-Whitney U test; proline, n = 15 mice for WT, 11 for HT, 14 for KO, ***P < 0.001, ns, not significant, Mann-Whitney U test). The error bars represent SEM.


( B ) Glycine / proline - induced currents for mouse SLC6A20A are dependent on sodium chloride , as shown by suppression of currents when sodium chloride is replaced with choline chloride or sodium gluconate . Each experimental condition was sequentially applied to HEK293T , as shown in Appendix Fig . S7B . indicated currents are average values from an ensemble of multiple ( ~ 20 ) in a single well . ( glycine , n = 21 for untransfected / blank , 17 for human SLC6A20 - V1 , 25 for human SLC6A20 - V2 , 33 for mSLC6A20A , 28 for mSLC6A20B * * * P < 0 . 001 ( relative to buffer not containing glycine ) , two - way ANOVA with Bonferroni ' s ; proline , n = 17 for untransfected , 23 for human SLC6A20 - V1 , 47 for SLC6A20 - V2 , 31 for mSLC6A20A , 31 for mSLC6A20B , * * * P < 0 . 001 , two - way ANOVA with Bonferroni ' s ) . error bars represent .

O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O 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 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 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 B-MEASURED_VAR 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) Glycine/proline-induced currents for mouse SLC6A20A are dependent on sodium chloride, as shown by the suppression of the currents when sodium chloride is replaced with choline chloride or sodium gluconate. Each experimental condition was sequentially applied to HEK293T cells, as shown in Appendix Fig. S7B. The indicated currents are average values from an ensemble of multiple (~20) cells in a single well. (glycine, n = 21 cells for untransfected/blank, 17 for human SLC6A20-V1, 25 for human SLC6A20-V2, 33 for mSLC6A20A, 28 for mSLC6A20B ***P < 0.001 (relative to buffer not containing glycine), two-way ANOVA with Bonferroni's test; proline, n = 17 cells for untransfected, 23 for human SLC6A20-V1, 47 for SLC6A20-V2, 31 for mSLC6A20A, and 31 for mSLC6A20B, ***P < 0.001, two-way ANOVA with Bonferroni's test). The error bars represent SEM.


( A - C ) Immunoblots for indicated in BAT , extensor digitorum longus ( EDL ) , epididymal adipose tissue ( eWAT ) heart from 10 ‐ to 12 ‐ month ( mo ) ‐ old control ( Con ) knock out ( KO ) mice . Arrows depict LC3 ‐ I II .

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(A-C) Immunoblots for indicated proteins in BAT, extensor digitorum longus (EDL), epididymal white adipose tissue (eWAT) and heart from 10‐ to 12‐month (mo)‐old control (Con) and knock out (KO) mice. Arrows depict LC3‐I and II.


( D ) ATG5 ATG7 mRNA in indicated tissues ( n = 4 )

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(D) ATG5 and ATG7 mRNA levels in indicated tissues (n=4)


( E ) immunoblots for indicated in spleen , liver , lung , kidney , mediobasal hypothalamus ( MBH ) , inguinal adipose tissue ( ingWAT ) perinephric fat ( pnWAT ) from 10 ‐ to 12 ‐ mo ‐ old Con KO mice .

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(E) immunoblots for indicated proteins in spleen, liver, lung, kidney, mediobasal hypothalamus (MBH), inguinal white adipose tissue (ingWAT) and perinephric fat (pnWAT) from 10‐ to 12‐mo‐old Con and KO mice.


( F ) weights ( wt ) of chow diet ( RD ) ‐ fed ( n = 6 - 29 ) , ( G ) high ‐ fat diet ( HFD ) ‐ fed Con KO mice at indicated ages ( n = 4 - 17 ) .

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(F) Body weights (wt) of chow diet (RD)‐fed (n=6-29), and (G) high‐fat diet (HFD)‐fed Con and KO mice at indicated ages (n=4-17).


( H ) Total fat lean mass of 4 - 7 mo RD ‐ fed Con KO mice ( n = 8 - 12 ) .

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(H) Total body fat and lean mass of 4-7 mo RD‐fed Con and KO mice (n=8-12).


( I ) BAT wt ( n = 4 - 7 ) , ( J ) gastrocnemius ( GA ) wt ( n = 5 - 7 ) , ( K ) soleus wt ( n = 4 - 7 ) , ( L ) eWAT wt ( n = 5 - 7 ) ( M ) visceral ( Visc ) subcutaneous ( Subc ) fat distribution in 10 ‐ mo ‐ old Con KO mice ( n = 4 ) . Values are mean ± s . e . , * P0 . 05 , * * P0 . 01 , * * * P0 . 001 .

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(I) BAT wt (n=4-7), (J) gastrocnemius (GA) wt (n=5-7), (K) soleus wt (n=4-7), (L) eWAT wt (n=5-7) and (M) visceral (Visc) and subcutaneous (Subc) body fat distribution in 10‐mo‐old Con and KO mice (n=4). Values are mean±s.e., *P0.05, **P0.01, ***P0.001.


( A - B ) mRNA for indicated genes in BAT ( n = 4 )

(A-B) mRNA for indicated genes in BAT (n=4)


( c ) mRNA for indicated genes in epididymal adipose tissue ( eWAT ) ( n = 4 )

(c) mRNA for the indicated genes in epididymal white adipose tissue (eWAT) (n=4)


( D ) immunoblots for indicated in BAT from 10 ‐ month ( mo ) ‐ old chow diet ( RD ) ‐ fed control ( Con ) knock out ( KO ) mice . Arrows depict isoforms .

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(D) immunoblots for indicated proteins in BAT from 10‐month (mo)‐old chow diet (RD)‐fed control (Con) and knock out (KO) mice. Arrows depict protein isoforms.


( E ) immunoblots for indicated in eWAT from 10 ‐ month ( mo ) ‐ old chow diet ( RD ) ‐ fed control ( Con ) knock out ( KO ) mice . Arrows depict isoforms .

(E) immunoblots for indicated proteins in eWAT from 10‐month (mo)‐old chow diet (RD)‐fed control (Con) and knock out (KO) mice. Arrows depict protein isoforms.


( F ) Electron ( × 10 , 000 magnification ) of BAT depicting mitochondria from 4 ‐ mo ‐ old Con KO mice . m , mitochondria ; LD , lipid droplet ; n , nucleus .

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(F) Electron micrographs ( × 10,000 magnification) of BAT depicting mitochondria from 4‐mo‐old Con and KO mice. m, mitochondria; LD, lipid droplet; n, nucleus.


( G ) mRNA ( n = 4 ) in BAT from 10 ‐ mo ‐ old Con KO

(G) mRNA levels (n=4) in BAT from 10‐mo‐old Con and KO mice


( H ) hematoxylin eosin ( HE ) ( I ) Sirius Red stains in BAT from 10 ‐ mo ‐ old Con KO mice ( n = 3 - 4 ) . Average adipocyte LD , LD number in BAT are shown . Scale bar , 50 μm .

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(H) hematoxylin and eosin (HE) and (I) Sirius Red stains in BAT from 10‐mo‐old Con and KO mice (n=3-4). Average adipocyte and LD size, and LD number in BAT are shown. Scale bar, 50 μm.


( J ) BAT mRNA from 4 ‐ mo ‐ old RD ‐ fed Con KO mice cold ‐ challenged for 75 min ( n = 3 ) . Values are mean ± s . e . * * * P0 . 001 .

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(J) BAT mRNA levels from 4‐mo‐old RD‐fed Con and KO mice cold‐challenged for 75 min (n=3). Values are mean±s.e. ***P0.001.


( A ) before ( Basal ) during cold exposure ( n = 7 - 8 ) in 10 ‐ month ( mo ) ‐ old chow diet ( RD ) ‐ fed control ( Con ) knock out ( KO )

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(A) Body temperature before (Basal) and during cold exposure (n=7-8) in 10‐month (mo)‐old chow diet (RD)‐fed control (Con) and knock out (KO) mice


( B ) Oxygen consumption ( VO2 ) , ( C ) carbon dioxide production ( VCO2 ) in 10 ‐ month ( mo ) ‐ old chow diet ( RD ) ‐ fed control ( Con ) knock out ( KO ) mice ( n = 4 ) .

(B) Oxygen consumption (VO2), (C) carbon dioxide production (VCO2) in 10‐month (mo)‐old chow diet (RD)‐fed control (Con) and knock out (KO) mice (n=4).


( D ) energy expenditure in 10 ‐ month ( mo ) ‐ old chow diet ( RD ) ‐ fed control ( Con ) knock out ( KO ) mice ( n = 4 )

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(D) energy expenditure in 10‐month (mo)‐old chow diet (RD)‐fed control (Con) and knock out (KO) mice (n=4)


( E ) VO2 , ( F ) VCO2 ( G ) energy expenditure in 10 ‐ mo ‐ old high ‐ fat diet ( HFD ) ‐ fed Con KO mice ( n = 3 - 5 ) .

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(E) VO2, (F) VCO2 and (G) energy expenditure in 10‐mo‐old high‐fat diet (HFD)‐fed Con and KO mice (n=3-5).


( H ) Ambulation ( AMB ) in 10 ‐ mo ‐ old RD ‐ fed ( n = 4 ) HFD ‐ fed Con KO mice ( n = 3 - 5 ) .

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(H) Ambulation (AMB) in 10‐mo‐old RD‐fed (n=4) and HFD‐fed Con and KO mice (n=3-5).


( I ) Hematoxylin eosin ( HE ) ‐ stained epididymal adipose tissue ( eWAT ) from 6 ‐ mo ‐ old RD ‐ fed Con KO mice . for is shown ( n = 3 ) . Scale bar , 50 μm .

(I) Hematoxylin and eosin (HE)‐stained epididymal white adipose tissue (eWAT) from 6‐mo‐old RD‐fed Con and KO mice. Quantification for cell size is shown (n=3). Scale bar, 50 μm.


( J ) Adβ3 mRNA in eWAT from 10 ‐ mo ‐ old RD ‐ fed Con KO mice ( n = 4 ) , in ( K ) inguinal ( ingWAT ) from 4 ‐ mo ‐ oldcold ‐ challenged RD ‐ fed Con KO mice ( n = 3 ) .

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(J) Adβ3 mRNA in eWAT from 10‐mo‐old RD‐fed Con and KO mice (n=4), and in (K) inguinal (ingWAT) from 4‐mo‐oldcold‐challenged RD‐fed Con and KO mice (n=3).


( L ) Serum ‐ free fatty acid ( FFA ) glycerol ( Gly ) from 4 ‐ to 6 ‐ mo ‐ old Con KO mice untreated ( basal ) or treated with isoproterenol ( Iso ) i . p . for 20 min ( n = 4 - 6 ) , ( M ) from 4 ‐ mo ‐ old RD ‐ fed cold ‐ challenged ( 75 min ) mice ( n = 3 - 4 ) . Values are mean ± s . e . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 .

O O 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 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

(L) Serum‐free fatty acid (FFA) and glycerol (Gly) from 4‐ to 6‐mo‐old Con and KO mice untreated (basal) or treated with isoproterenol (Iso) i.p. for 20 min (n=4-6), and (M) from 4‐mo‐old RD‐fed cold‐challenged (75 min) mice (n=3-4). Values are mean±s.e. *P0.05, **P0.01, ***P0.001.


( A , B ) Tmem26 tbx1 mRNA in epididymal eWAT , ( C , D ) ingWAT from Atg7Flox / Floxmice ( 4 months ( mo ) ) injected in BAT with empty ( Con AdV ) or Cre ‐ expressing ( Cre AdV ) adenoviruses cold ‐ exposed or not ( basal ) for 75 min ( n = 4 - 5 ) .

O O O O O B-MEASURED_VAR I-MEASURED_VAR 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 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

(A,B) Tmem26 and tbx1 mRNA levels in epididymal eWAT, and (C,D) ingWAT from Atg7Flox/Floxmice (4 months (mo)) injected in BAT with empty (Con AdV) or Cre‐expressing (Cre AdV) adenoviruses and cold‐exposed or not (basal) for 75 min (n=4-5).


( E , F ) hematoxylin eosin ( HE ) ‐ stained ingWAT section from Atg7Flox / Flox mice ( 4 months ( mo ) ) injected in BAT with empty ( Con AdV ) or Cre ‐ expressing ( Cre AdV ) adenoviruses cold ‐ exposed or not ( basal ) for 75 min ( n = 4 - 5 ) .

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

(E,F) hematoxylin and eosin (HE)‐stained ingWAT section from Atg7Flox/Flox mice (4 months (mo)) injected in BAT with empty (Con AdV) or Cre‐expressing (Cre AdV) adenoviruses and cold‐exposed or not (basal) for 75 min (n=4-5).


( G ) mRNA for indicated genes in ingWAT from 4 ‐ mo ‐ old cold ‐ exposed Con KO mice ( n = 4 ) .

O O O 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) mRNA for indicated genes in ingWAT from 4‐mo‐old cold‐exposed Con and KO mice (n=4).


( H , I ) mRNA in eWAT from 10 ‐ mo ‐ old Con KO mice ( n = 4 ) .

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

(H,I) mRNA levels in eWAT from 10‐mo‐old Con and KO mice (n=4).


( J ) β ‐ oxidation in eWAT ingWAT from 4 ‐ mo ‐ old Con KO mice ( n = 4 ) .

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) β‐oxidation in eWAT and ingWAT from 4‐mo‐old Con and KO mice (n=4).


( K ) mRNA in soleus from 4 ‐ mo ‐ old cold ‐ exposed Con KO mice ( n = 3 - 6 ) .

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

(K) mRNA levels in soleus from 4‐mo‐old cold‐exposed Con and KO mice (n=3-6).


( M ) BAT from 4 ‐ mo ‐ old Con KO mice ( n = 4 ) . Values are mean ± s . e . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 .

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

(M) BAT from 4‐mo‐old Con and KO mice (n=4). Values are mean±s.e. *P0.05, **P0.01, ***P0.001.


( A ) Blood glucose in 6 ‐ to 10 ‐ month ( mo ) ‐ old mice ( n = 12 - 15 ) , ( B ) serum insulin in 10 ‐ mo ‐ old fed control ( Con ) KO mice ( n = 6 ) .

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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(A) Blood glucose in 6‐ to 10‐month (mo)‐old mice (n=12-15), and (B) serum insulin levels in 10‐mo‐old fed control (Con) and KO mice (n=6).


( C ) Glucose tolerance in 10 ‐ mo ‐ old chow diet ( RD ) ‐ fed ( n = 5 ) , ( D ) in 10 ‐ to 12 ‐ mo ‐ old high ‐ fat diet ( HFD ) ‐ fed Con KO mice ( n = 4 - 9 ) .

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

(C) Glucose tolerance tests in 10‐mo‐old chow diet (RD)‐fed (n=5), and (D) in 10‐ to 12‐mo‐old high‐fat diet (HFD)‐fed Con and KO mice (n=4-9).


( E ) Insulin tolerance in 10 ‐ mo ‐ old RD ‐ fed Con KO mice ( n = 5 ) .

(E) Insulin tolerance test in 10‐mo‐old RD‐fed Con and KO mice (n=5).


( F ) Immunoblots for indicated in skeletal muscle from 10 ‐ mo ‐ old Con KO mice .

(F) Immunoblots for indicated proteins in skeletal muscle from 10‐mo‐old Con and KO mice.


( G ) Hematoxylin eosin ( HE ) ‐ stained gastrocnemius ( GA ) sections from 6 ‐ mo ‐ old Con KO mice . Myofiber cross ‐ sectional ( μm2 ) is shown ( n = 3 ) .

(G) Hematoxylin and eosin (HE)‐stained gastrocnemius (GA) sections from 6‐mo‐old Con and KO mice. Myofiber cross‐sectional area (μm2) is shown (n=3).


( H ) Expression of MuRF ‐ 1 Atrogin ‐ 1 in GA TA from 10 ‐ mo ‐ old Con KO mice ( n = 4 )

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

(H) Expression of MuRF‐1 and Atrogin‐1 in GA and TA from 10‐mo‐old Con and KO mice (n=4)


( I ) myogenic genes in GA from 10 ‐ mo ‐ old Con KO mice ( n = 6 ) .

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

(I) myogenic genes in GA from 10‐mo‐old Con and KO mice (n=6).


( J ) Immunoblots for indicated in tissues from 4 ‐ mo ‐ old Con KO mice ( n = 4 ) .

(J) Immunoblots for indicated proteins in tissues from 4‐mo‐old Con and KO mice (n=4).


( K ) mRNA for IRS1 , ( L ) IRS2 in epididymal adipose tissue ( eWAT ) GA from 10 ‐ mo ‐ old Con KO mice ( n = 6 ) .

O O O O O B-MEASURED_VAR 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

(K) mRNA for IRS1, (L) IRS2 in epididymal white adipose tissue (eWAT) and GA from 10‐mo‐old Con and KO mice (n=6).