A ) Representative mEPSC traces recorded from control and PTX3 - treated ( 1µg / ml ; 48hrs ) neurons . B ) mEPSC frequency quantitation ( Hz , Ctr = 0 . 618 ± 0 . 069 ; PTX3 = 1 . 991 ± 0 . 313 ; Number of neurons : Ctr = 22 , PTX3 = 16 ; 3 independent experiments , Mann - Whitney test . Data are presented as a distribution plus mean ± SEM ) . C ) mEPSC amplitude quantitation and cumulative probability distribution of mEPSC amplitudes ( pA , Ctr = 12 . 76 ± 0 . 813 ; PTX3 = 16 . 07 ± 0 . 709 . Number of neurons : Ctr = 22 , PTX3 = 16 ; 3 independent experiments , Mann - Whitney test . Data are presented as a distribution plus mean ± SEM . Cumulative probability distributions are analyzed by Kolmogorov - Smirnov

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 mEPSC traces recorded from control and PTX3-treated (1µg/ml; 48hrs) neurons. B) mEPSC frequency quantitation (Hz, Ctr=0.618±0.069; PTX3=1.991±0.313; Number of neurons: Ctr=22, PTX3=16; 3 independent experiments, Mann-Whitney test. Data are presented as a distribution plus mean±SEM). C) mEPSC amplitude quantitation and cumulative probability distribution of mEPSC amplitudes (pA, Ctr=12.76±0.813; PTX3=16.07±0.709. Number of neurons: Ctr=22, PTX3=16; 3 independent experiments, Mann-Whitney test. Data are presented as a distribution plus mean±SEM. Cumulative probability distributions are analyzed by Kolmogorov-Smirnov test


D ) Representative images showing 14DIV neurons stained for surface AMPAR ( GluA , green ) , the presynaptic protein Bassoon ( blue ) and tubulin ( red ) in the different tested conditions . Arrowheads point to postsynaptic GluA clusters . Inset : Example of surface synaptic AMPARs cluster ( GluA & Bsn ) . Scale bar : 5 µm . E ) Quantification of the surface synaptic AMPARs ( GluA & Bsn ) normalized to the total number of Bsn shows a statistically significant increase after TTX or PTX3 exposure ( Ctr = 1 ± 0 . 051 , TTX = 1 . 512 ± 0 . 080 , PTX3 = 1 . 294 ± 0 . 081 , PTX3 heat - inactivated = 0 . 959 ± 0 . 044 ; number of fields examined : 27 , 36 , 37 , 22 , 19 respectively ; one - way ANOVA analysis of variance , p < 0 . 0001 , followed by post hoc Tukey test for multiple comparison as indicated in figure ; 3 independent experiments , data are presented as normalized mean values ± SEM ) . F ) 1hr of PTX3 administration is not sufficient to elicit an increase in synaptic GluA content ( Ctr : 1 ± 0 . 065 , PTX3 : 1 . 023 ± 0 . 068 ; number of fields : Ctr = 26 , PTX3 = 26 ; unpaired t - test , 3 independent experiments , data are presented as normalized mean values ± 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 B-CONTROLLED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images showing 14DIV neurons stained for surface AMPAR (GluA, green), the presynaptic protein Bassoon (blue) and tubulin (red) in the different tested conditions. Arrowheads point to postsynaptic GluA clusters. Inset: Example of surface synaptic AMPARs cluster (GluA&Bsn). Scale bar: 5 µm. E) Quantification of the surface synaptic AMPARs (GluA&Bsn) normalized to the total number of Bsn shows a statistically significant increase after TTX or PTX3 exposure (Ctr=1±0.051, TTX=1.512±0.080, PTX3=1.294±0.081, PTX3 heat-inactivated=0.959±0.044; number of fields examined: 27, 36, 37, 22, 19 respectively; one-way ANOVA analysis of variance, p<0.0001, followed by post hoc Tukey test for multiple comparison as indicated in figure; 3 independent experiments, data are presented as normalized mean values±SEM). F) 1hr of PTX3 administration is not sufficient to elicit an increase in synaptic GluA content (Ctr: 1±0.065, PTX3: 1.023±0.068; number of fields: Ctr=26, PTX3=26; unpaired t-test, 3 independent experiments, data are presented as normalized mean values±SEM)


G ) Representative mEPSC traces recorded from control , C - term PTX3 fragment or N - term PTX3 fragment - treated cultures . H ) mEPSC frequency quantitation ( Hz , Ctr = 0 . 668 ± 0 . 078 ; C - term = 0 . 523 ± 0 . 056 ; N - term = 1 . 228 ± 0 . 121 . Number of neurons : Ctr = 23 , C - term = 10 , N - term = 23 ; 3 independent experiments . Kruskall - Wallis test , p < 0 . 0001 followed by Dunn " s test as indicated , data are presented as a distribution plus mean ± SEM ) . I ) mEPSC amplitude quantitation ( pA , Ctr = 16 . 81 ± 0 . 78 ; C - term = 15 . 79 ± 1 . 37 ; N - term = 19 . 78 ± 0 . 87 . Number of neurons : Ctr = 23 , C - term = 10 , N - term = 23 ; 3 independent experiments . One - way ANOVA analysis of variance , p = 0 . 013 followed by post hoc Tukey test as indicated in figure , data are presented as a distribution plus mean ± SEM ) and cumulative probability distribution of mEPSC amplitudes ( Kolmogorov -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative mEPSC traces recorded from control, C-term PTX3 fragment or N-term PTX3 fragment-treated cultures. H) mEPSC frequency quantitation (Hz, Ctr=0.668±0.078; C-term=0.523±0.056; N-term=1.228±0.121. Number of neurons: Ctr=23, C-term=10, N-term=23; 3 independent experiments. Kruskall-Wallis test, p<0.0001 followed by Dunn"s test as indicated, data are presented as a distribution plus mean±SEM). I) mEPSC amplitude quantitation (pA, Ctr=16.81±0.78; C-term=15.79±1.37; N-term=19.78±0.87. Number of neurons: Ctr=23, C-term=10, N-term=23; 3 independent experiments. One-way ANOVA analysis of variance, p=0.013 followed by post hoc Tukey test as indicated in figure, data are presented as a distribution plus mean±SEM) and cumulative probability distribution of mEPSC amplitudes (Kolmogorov-Smirnov


J ) Representative images of 14DIV neurons stained for surface AMPARs ( GluA , green ) , the presynaptic protein Bassoon ( blue ) and tubulin ( red ) in the different tested conditions . Arrowheads point to postsynaptic GluA clusters . Scale bar : 5µm . K ) Quantification of the surface synaptic AMPARs normalized to the total number of Bsn shows an increase upon N - terminal peptide application , but not upon C - terminal peptide exposure . TTX is used as positive control ( Ctr = 1 . 000 ± 0 . 066 . TTX = 1 . 395 ± 0 . 113 , C - term = 0 . 717 ± 0 . 047 , N - term = 1 . 349 ± 0 . 069 . Number of fields examined : 55 , 31 , 18 , 54 , respectively ; one - way ANOVA analysis of variance , p < 0 . 0001 followed by post hoc Tukey test as indicated in figure ; at least 3 independent experiments , data are presented as normalized mean values ± 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 B-CONTROLLED_VAR 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

J) Representative images of 14DIV neurons stained for surface AMPARs (GluA, green), the presynaptic protein Bassoon (blue) and tubulin (red) in the different tested conditions. Arrowheads point to postsynaptic GluA clusters. Scale bar: 5µm. K) Quantification of the surface synaptic AMPARs normalized to the total number of Bsn shows an increase upon N-terminal peptide application, but not upon C-terminal peptide exposure. TTX is used as positive control (Ctr=1.000±0.066. TTX=1.395±0.113, C-term=0.717±0.047, N-term=1.349±0.069. Number of fields examined: 55, 31, 18, 54, respectively; one-way ANOVA analysis of variance, p<0.0001 followed by post hoc Tukey test as indicated in figure; at least 3 independent experiments, data are presented as normalized mean values±SEM).


A ) Low and high magnification images of control and PTX3 - treated neurons stained for the PNN main component , aggrecan ( red ) , the synaptic proteins PSD95 ( green ) and Bsn ( blue ) . Scale bar :

O O O O O O O O O O O O O O O O O O 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) Low and high magnification images of control and PTX3-treated neurons stained for the PNN main component, aggrecan (red), the synaptic proteins PSD95 (green) and Bsn (blue). Scale bar: 5µm


B - D ) PTX3 application induces a remodeling of the PNN in culture , as assessed by the increase mean intensity and integrated density value of the synapse - co - localizing aggrecan signal whereas no difference in the total area of aggrecan is evident ( integrated density : Ctr = 1 ± 0 . 103 , PTX3 = 1 . 874 ± 0 . 197 ; mean intensity : Ctr = 1 . 000 ± 0 . 088 , PTX3 = 1 . 556 ± 0 . 107 ; total area : Ctr = 1 ± 0 . 179 , PTX3 = 0 . 982 ± 0 . 149 . Number of fields examined : 26 Ctr , 22 PTX3 ; Mann - Whitney test ; 3 independent experiments , data are presented as normalized mean values ± 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

B-D) PTX3 application induces a remodeling of the PNN in culture, as assessed by the increase mean intensity and integrated density value of the synapse-co-localizing aggrecan signal whereas no difference in the total area of aggrecan is evident (integrated density: Ctr=1±0.103, PTX3=1.874±0.197; mean intensity: Ctr=1.000±0.088, PTX3=1.556±0.107; total area: Ctr=1±0.179, PTX3=0.982±0.149. Number of fields examined: 26 Ctr, 22 PTX3; Mann-Whitney test; 3 independent experiments, data are presented as normalized mean values±SEM)


E ) Overnight treatment with hyaluronidase destroys PNN as shown by immunofluorescence for aggrecan ( red ) , DAPI ( cyan ) and βIII - tubulin ( green ) and confocal analysis . Scale bar :

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

E) Overnight treatment with hyaluronidase destroys PNN as shown by immunofluorescence for aggrecan (red), DAPI (cyan) and βIII-tubulin (green) and confocal analysis. Scale bar: 20µm


F ) Representative images showing 14DIV neurons stained for surface AMPARs ( GluA , green ) , the presynaptic protein Bassoon ( blue ) and tubulin ( red ) in the different tested conditions . Arrowheads point to postsynaptic GluA clusters . Scale bar :

O O O O O O O O O O O O O O O O O O O O O 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 images showing 14DIV neurons stained for surface AMPARs (GluA, green), the presynaptic protein Bassoon (blue) and tubulin (red) in the different tested conditions. Arrowheads point to postsynaptic GluA clusters. Scale bar: 5µm


G ) HAse treatment blocks PTX3 - induced synaptic surface AMPA receptors clustering ( Ctr = 1 . 000 ± 0 . 075 , PTX3 = 1 . 425 ± 0 . 088 , PTX3 + HAse = 0 . 961 ± 0 . 053 , HAse = 1 . 080 ± 0 . 087 . Number of fields examined : 37 , 23 , 53 , 28 respectively ; Kruskall - Wallis test , p = 0 . 0004 followed by post hoc Tukey test as indicated in figure ; 3 independent experiments , data are presented as normalized mean value ± SEM )

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

G) HAse treatment blocks PTX3-induced synaptic surface AMPA receptors clustering (Ctr=1.000±0.075, PTX3=1.425±0.088, PTX3+HAse=0.961±0.053, HAse=1.080±0.087. Number of fields examined: 37, 23, 53, 28 respectively; Kruskall-Wallis test, p=0.0004 followed by post hoc Tukey test as indicated in figure; 3 independent experiments, data are presented as normalized mean value±SEM)


H ) Representative images showing 14DIV WT and TSG6 KO neurons stained for surface AMPARs ( GluA , green ) , the presynaptic protein Bassoon ( blue ) and tubulin ( red ) in the different tested conditions . Arrowheads point to postsynaptic GluA clusters . Scale bar :

O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images showing 14DIV WT and TSG6 KO neurons stained for surface AMPARs (GluA, green), the presynaptic protein Bassoon (blue) and tubulin (red) in the different tested conditions. Arrowheads point to postsynaptic GluA clusters. Scale bar: 5µm


I ) Synaptic surface GluA quantitation showing no effect of PTX3 treatment in TSG6 KO cultures . On the contrary WT cultures ( from littermates ) display increased surface GluA & Bsn / Bsn upon PTX3 treatment . A significant enhancement of surface GluA receptors was induced by TTX in both TSG6 KO and WT cultures ( WT = 1 ± 0 . 06 ; WT + PTX3 = 1 . 389 ± 0 . 113 ; WT + TTX = 1 . 698 ± 0 . 109 ; Number of fields examined : 40 , 39 , 32 respectively ; Kruskall - Wallis test p < 0 . 0001 followed by post hoc Dunn " s test . TSG6 KO = 1 . 000 ± 0 . 047 ; TSG6 KO + PTX3 = 0 . 979 ± 0 . 048 ; TSG6 KO + TTX = 1 . 363 ± 0 . 077 . Number of fields examined : 54 , 57 , 29 respectively ; one - way ANOVA analysis of variance , p < 0 . 0001 followed by post hoc Tukey test as indicated in figure . n = 3 independent experiments , data are presented as normalized mean

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

I) Synaptic surface GluA quantitation showing no effect of PTX3 treatment in TSG6 KO cultures. On the contrary WT cultures (from littermates) display increased surface GluA&Bsn/Bsn upon PTX3 treatment. A significant enhancement of surface GluA receptors was induced by TTX in both TSG6 KO and WT cultures (WT=1±0.06; WT+PTX3=1.389±0.113; WT+TTX=1.698±0.109; Number of fields examined: 40, 39, 32 respectively; Kruskall-Wallis test p<0.0001 followed by post hoc Dunn"s test. TSG6 KO=1.000±0.047; TSG6 KO+PTX3=0.979±0.048; TSG6 KO+TTX=1.363±0.077. Number of fields examined: 54, 57, 29 respectively; one-way ANOVA analysis of variance, p<0.0001 followed by post hoc Tukey test as indicated in figure. n=3 independent experiments, data are presented as normalized mean values


J ) Representative mEPSC traces recorded from WT and TSG6 KO littermates cultures treated or not with PTX3 ( 1μg / ml for 48hrs ) . K ) On the contrary of WT cultures , mEPSC frequency quantitation shows no increase in frequency in TSG6 KO cultures when treated with PTX3 ( Normalized Frequency , WT Ctr : 1 . 000 ± 0 . 074 ; Wt + PTX3 = 1 . 912 ± 0 . 289 ; TSG6 KO Ctr = 1 ± 0 . 079 ; TSG6 KO + PTX3 = 1 . 226 ± 0 . 096 ; 22 , 20 , 30 , 24 cells respectively , 6 TSG6 KO mice and 4 WT littermates . One way ANOVA test p = 0 . 002 followed by post hoc Dunn ' s test , data are presented as normalized mean ± SEM plus the distribution ) . L ) Cumulative probability plot of mEPSC amplitude and average mEPSC amplitude quantitation ( inset ) showing no difference in amplitude ( WT Ctr : 1 . 000 ± 0 . 034 , Wt + PTX3 = 1 . 069 ± 0 . 046 , TSG6 KO Ctr = 1 . 000 ± 0 . 028 ; TSG6 KO + PTX3 = 1 . 074 ± 0 . 67 ; 22 , 20 , 30 , 24 cells respectively , 6 TSG6 KO mice and 4 WT littermates . One way ANOVA test p = 0 . 817 followed by post hoc Dunn ' s test , data are presented as normalized mean ± SEM plus the distribution . Cumulative distribution is analyzed by Kolmogorov - Smirnov 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative mEPSC traces recorded from WT and TSG6 KO littermates cultures treated or not with PTX3 (1μg/ml for 48hrs). K) On the contrary of WT cultures, mEPSC frequency quantitation shows no increase in frequency in TSG6 KO cultures when treated with PTX3(Normalized Frequency, WT Ctr: 1.000±0.074; Wt+PTX3=1.912±0.289; TSG6 KO Ctr=1±0.079; TSG6 KO+PTX3=1.226±0.096; 22, 20, 30, 24 cells respectively, 6 TSG6 KO mice and 4 WT littermates. One way ANOVA test p=0.002 followed by post hoc Dunn's test, data are presented as normalized mean±SEM plus the distribution). L) Cumulative probability plot of mEPSC amplitude and average mEPSC amplitude quantitation (inset) showing no difference in amplitude (WT Ctr: 1.000±0.034, Wt+PTX3=1.069±0.046, TSG6 KO Ctr =1.000±0.028; TSG6 KO+PTX3=1.074±0.67; 22, 20, 30, 24 cells respectively, 6 TSG6 KO mice and 4 WT littermates. One way ANOVA test p=0.817 followed by post hoc Dunn's test, data are presented as normalized mean±SEM plus the distribution. Cumulative distribution is analyzed by Kolmogorov-Smirnov test).


A ) Representative images showing 14DIV neurons stained for surface AMPARs ( GluA , green ) , the presynaptic protein Bassoon ( blue ) and tubulin ( red ) in the different tested conditions . Arrowheads point to postsynaptic GluA clusters . Scale bar :

A) Representative images showing 14DIV neurons stained for surface AMPARs (GluA, green), the presynaptic protein Bassoon (blue) and tubulin (red) in the different tested conditions. Arrowheads point to postsynaptic GluA clusters. Scale bar: 5µm


B ) Blocking β1 integrins activity by using the specific anti - β1 Integrin monoclonal antibody prevents the PTX3 - induced postsynaptic AMPARs recruitment ( Ctr = 1 . 000 ± 0 . 074 , PTX3 = 1 . 504 ± 0 . 098 , αCD29 + PTX3 = 0 . 766 ± 0 . 097 , αCD29 = 0 . 810 ± 0 . 108 . Number of fields examined : 37 , 33 , 23 , 24 respectively ; one way ANOVA analysis of variance , p < 0 . 0001 followed by post hoc Tukey test ; 3 independent experiments , data are presented as normalized mean values ± 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

B) Blocking β1 integrins activity by using the specific anti-β1 Integrin monoclonal antibody prevents the PTX3-induced postsynaptic AMPARs recruitment (Ctr=1.000±0.074, PTX3=1.504±0.098, αCD29+PTX3=0.766±0.097, αCD29=0.810±0.108. Number of fields examined: 37, 33, 23, 24 respectively; one way ANOVA analysis of variance, p<0.0001 followed by post hoc Tukey test; 3 independent experiments, data are presented as normalized mean values±SEM)


C ) Western blotting analysis of p - ERK levels on lysates from control and PTX3 - treated neurons upon 30 minutes stimulation ( Ctr = 1 ± 0 ; PTX3 = 1 . 453 ± 0 . 195 , 6 independent experiments , unpaired t - test , data are presented normalized on control and 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

C) Western blotting analysis of p-ERK levels on lysates from control and PTX3-treated neurons upon 30 minutes stimulation (Ctr=1±0; PTX3=1.453±0.195, 6 independent experiments, unpaired t-test, data are presented normalized on control and as mean±SEM)


D ) Representative mEPSC traces recorded from the indicated experimental conditions . E ) mEPSC frequency quantitation showing that pre - incubation with PD98059 ( 30μM ) completely prevents the PTX3 - dependent increase of mEPSC frequency ( Hz , Ctr = 0 . 911 ± 0 . 104 ; PTX3 = 1 . 748 ± 0 . 273 ; PTX3 + PD = 0 . 518 ± 0 . 055 ; PD = 0 . 491 ± 0 . 104 . Number of neurons : Ctr = 14 , PTX3 = 14 , PTX3 + PD = 13 ; PD = 9 ; 3 independent experiments . One - way ANOVA analysis of variance , p < 0 . 0001 followed by post hoc Tukey test as indicated in figure . Data are presented as a distribution plus mean ± SEM ) . F ) inset : average mEPSC amplitude quantitation ( pA ) , Ctr = 14 . 19 ± 0 . 70 ; PTX3 = 19 . 09 ± 1 . 71 ; PTX3 + PD = 14 . 31 ± 0 . 70 , PD = 16 . 51 ± 1 . 17 . Number of neurons : Ctr = 14 , PTX3 = 14 , PTX3 + PD = 13 ; PD = 9 ; 3 independent experiments . Kruskall - Wallis test , p = 0 . 015 followed by Dunn " s test as indicated in figure , data are presented as a distribution plus mean ± SEM . Cumulative probability plot of mEPSC amplitudes are analyzed with Kolmogorov - Smirnov test : Ctr vs PTX3 : p < 0 . 0001 , D = 0 . 183 , PTX3 vs PTX3 + PD : p < 0 . 0001 , D = 0 . 251 , PTX3 vs PD : p < 0 . 0001 , D =

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 B-CONTROLLED_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

D) Representative mEPSC traces recorded from the indicated experimental conditions. E) mEPSC frequency quantitation showing that pre-incubation with PD98059 (30μM) completely prevents the PTX3-dependent increase of mEPSC frequency (Hz, Ctr=0.911±0.104; PTX3=1.748±0.273; PTX3+PD=0.518±0.055; PD=0.491±0.104. Number of neurons: Ctr=14, PTX3=14, PTX3+PD=13; PD=9; 3 independent experiments. One-way ANOVA analysis of variance, p<0.0001 followed by post hoc Tukey test as indicated in figure. Data are presented as a distribution plus mean±SEM). F) inset: average mEPSC amplitude quantitation (pA), Ctr=14.19±0.70; PTX3=19.09±1.71; PTX3+PD=14.31±0.70, PD=16.51±1.17. Number of neurons: Ctr=14, PTX3=14, PTX3+PD=13; PD=9; 3 independent experiments. Kruskall-Wallis test, p=0.015 followed by Dunn"s test as indicated in figure, data are presented as a distribution plus mean±SEM. Cumulative probability plot of mEPSC amplitudes are analyzed with Kolmogorov-Smirnov test: Ctr vs PTX3: p<0.0001, D=0.183, PTX3 vs PTX3+PD: p<0.0001, D=0.251, PTX3 vs PD: p<0.0001, D=0


G ) Representative images showing 14DIV neurons stained for surface AMPARs ( GluA , green ) , the presynaptic protein Bassoon ( blue ) and tubulin ( red ) in the different tested conditions . Arrowheads point to postsynaptic GluA clusters . Scale bar :

G) Representative images showing 14DIV neurons stained for surface AMPARs (GluA, green), the presynaptic protein Bassoon (blue) and tubulin (red) in the different tested conditions. Arrowheads point to postsynaptic GluA clusters. Scale bar: 5µm


H ) Quantification of the surface synaptic AMPARs normalized to the total number of Bsn [ ( GluA & Bsn ) / Bsn ] shows a statistically significant increase after PTX3 exposure , while pre - incubation with PD98059 prevents the PTX3 - dependent increase ( Ctr = 1 ± 0 . 042 , PTX3 = 1 . 499 ± 0 . 121 , PTX3 + PD = 1 . 093 ± 0 . 060 , PD = 1 . 017 ± 0 . 117 ; number of fields examined : 49 , 15 , 15 , 20 respectively ; one - way ANOVA analysis of variance , p = 0 . 0002 followed by post hoc Tukey test as indicated in figure ; at least 3 independent experiments , data are presented as normalized mean values ± 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 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 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

H) Quantification of the surface synaptic AMPARs normalized to the total number of Bsn [(GluA&Bsn)/Bsn] shows a statistically significant increase after PTX3 exposure, while pre-incubation with PD98059 prevents the PTX3-dependent increase (Ctr=1±0.042, PTX3=1.499±0.121, PTX3+PD=1.093±0.060, PD=1.017±0.117; number of fields examined: 49, 15, 15, 20 respectively; one-way ANOVA analysis of variance, p=0.0002 followed by post hoc Tukey test as indicated in figure; at least 3 independent experiments, data are presented as normalized mean values±SEM)


I ) Quantification of surface synaptic GluA upon PTX3 application and simultaneous inhibition of β1 integrin and ERK1 / 2 signaling pathways reveals no additive effects with respect to single αCD29 or PD applications ( Ctr = 1 ± 0 . 061 ; PTX3 = 1 . 828 ± 0 . 151 ; αCD29 + PD + PTX3 = 0 . 766 ± 0 . 070 ; PTX3 + αCD29 = 1 . 043 ± 0 . 073 ; PTX3 + PD = 1 . 016 ± 0 . 061 ; PD = 0 . 969 ± 0 . 090 ; number of fields examined : 29 , 31 , 30 , 23 , 14 , 33 respectively ; one - way ANOVA analysis of variance , p < 0 . 0001 followed by post hoc Dunn ' s test as indicated in figure ; at least 3 independent experiments , data are presented as normalized mean values ± 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 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 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-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 O O 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) Quantification of surface synaptic GluA upon PTX3 application and simultaneous inhibition of β1 integrin and ERK1/2 signaling pathways reveals no additive effects with respect to single αCD29 or PD applications (Ctr=1±0.061; PTX3=1.828±0.151; αCD29+PD+PTX3=0.766±0.070; PTX3+αCD29=1.043±0.073; PTX3+PD=1.016±0.061; PD=0.969±0.090; number of fields examined: 29, 31, 30, 23, 14, 33 respectively; one-way ANOVA analysis of variance, p<0.0001 followed by post hoc Dunn's test as indicated in figure; at least 3 independent experiments, data are presented as normalized mean values±SEM).


A ) PTX3 binds TSP1 and TSP - 2 but not TSP - 4 . Different amounts of human recombinant PTX3 were incubated in microplate wells coated with purified human TSP1 or recombinant TSP - 2 and TSP - 4 . Binding is reported as Absorbance at 450 nm ( Mean ± SD ) . Data are from one experiment out of three

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) PTX3 binds TSP1 and TSP-2 but not TSP-4. Different amounts of human recombinant PTX3 were incubated in microplate wells coated with purified human TSP1 or recombinant TSP-2 and TSP-4. Binding is reported as Absorbance at 450 nm (Mean±SD). Data are from one experiment out of three performed


B ) N - terminal PTX3 binds TSP1 . Binding of PTX3 N - term domain was performed on TSP1 immobilized on plastic wells . Data are reported as Absorbance at 450 nm ( Mean ± SD ) and are representative of one out of two experiments

O O O O O O O O O O O O O O O O O O O O O O O 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) N-terminal PTX3 binds TSP1. Binding of PTX3 N-term domain was performed on TSP1 immobilized on plastic wells. Data are reported as Absorbance at 450 nm (Mean±SD) and are representative of one out of two experiments performed


D ) PTX3 binds the C - term proteolytic fragment of TSP1 . TSP1 and its fragments C - term , N - term , and Ca - 1 ( type III repeats ) were immobilized in plastic wells ( 5µg / ml ) and binding of PTX3 is reported as Absorbance at 450 nm ( Mean ± SD ) . Data refers to one out of two experiments performed with similar

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) PTX3 binds the C-term proteolytic fragment of TSP1. TSP1 and its fragments C-term, N-term, and Ca-1 (type III repeats) were immobilized in plastic wells (5µg/ml) and binding of PTX3 is reported as Absorbance at 450 nm (Mean±SD). Data refers to one out of two experiments performed with similar results


E ) PTX3 and its N - terminal domain bind TSP1 C - terminal globular domain . 50 nM of P123 - 1 ( type I ' properdin ' repeats ) , E123 - 1 ( Type II EGF repeats ) , E123CaG - 1 ( type II repeats plus type III repeats and globular C terminus ) and TSP1 were immobilized in plastic well . Binding with PTX3 or N - terminal domain ( both at 220 nM ) was analyzed . Data are reported as Absorbance at 450 nm ( Mean ± SD ) and refers to one out of two experiments performed with similar

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) PTX3 and its N-terminal domain bind TSP1 C-terminal globular domain. 50 nM of P123-1 (type I 'properdin' repeats), E123-1 (Type II EGF repeats), E123CaG-1 (type II repeats plus type III repeats and globular C terminus) and TSP1 were immobilized in plastic well. Binding with PTX3 or N-terminal domain (both at 220 nM) was analyzed. Data are reported as Absorbance at 450 nm (Mean±SD) and refers to one out of two experiments performed with similar results


F ) Representative images of 14DIV control and PTX3 - treated cultures stained for the presynaptic marker bassoon ( blue ) and the microtubule protein tubulin ( red ) . Scale bar : 10μm . G ) Quantification of synaptic density ( Bsn / μm ) in the different experimental conditions ( Ctr = 0 . 385 ± 0 . 029 , PTX3 = 0 . 407 ± 0 . 032 , TSP1 = 0 . 721 ± 0 . 039 , PTX3 + TSP1 = 0 . 707 ± 0 . 037 , E123 = 0 . 764 ± 0 . 047 , PTX3 + E123 = 0 . 719 ± 0 . 041 . Number of fields examined : 86 , 80 , 75 , 72 , 46 , 55 respectively ; Kruskall - Wallis test , p < 0 . 0001 followed by Dunn " s test as indicated in figure ; at least 3 independent experiments , data are presented as mean

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images of 14DIV control and PTX3-treated cultures stained for the presynaptic marker bassoon (blue) and the microtubule protein tubulin (red). Scale bar: 10μm. G) Quantification of synaptic density (Bsn/μm) in the different experimental conditions (Ctr=0.385±0.029, PTX3=0.407±0.032, TSP1=0.721±0.039, PTX3+TSP1=0.707±0.037, E123=0.764±0.047, PTX3+E123=0.719±0.041. Number of fields examined: 86, 80, 75, 72, 46, 55 respectively; Kruskall-Wallis test, p<0.0001 followed by Dunn"s test as indicated in figure; at least 3 independent experiments, data are presented as mean values


H ) Representative images showing 14DIV neurons stained for surface AMPAR ( GluA , green ) , the presynaptic protein Bassoon ( blue ) and tubulin ( red ) in the different tested conditions . Arrowheads point to postsynaptic GluA clusters . Scale bar : 5µm . I ) Quantification of the synaptic surface GluA density ( number of sGluA puncta / μm ) in the different tested conditions showing that GluA density increases in neuronal cultures treated with PTX3 or E123 + PTX3 but not in cultures treated with the full length TSP1 + PTX3 ( Ctr = 1 . 000 ± 0 . 055 ; PTX3 = 1 . 367 ± 0 . 074 ; TSP1 = 1 . 04 ± 0 . 061 ; PTX3 + TSP1 = 0 . 968 ± 0 . 076 ; E123 = 0 . 971 ± 0 . 063 ; PTX3 + E123 = 1 . 613 ± 0 . 077 . Number of dendrites examined : 103 , 80 , 88 , 57 , 51 , 62 respectively ; Kruskall - Wallis test , p < 0 . 0001 followed by Dunn ' s test as indicated in figure ; at least 3 independent experiments , data are presented as normalized mean values ± 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

H) Representative images showing 14DIV neurons stained for surface AMPAR (GluA, green), the presynaptic protein Bassoon (blue) and tubulin (red) in the different tested conditions. Arrowheads point to postsynaptic GluA clusters. Scale bar: 5µm. I) Quantification of the synaptic surface GluA density (number of sGluA puncta/μm) in the different tested conditions showing that GluA density increases in neuronal cultures treated with PTX3 or E123+PTX3 but not in cultures treated with the full length TSP1+PTX3 (Ctr=1.000±0.055; PTX3=1.367±0.074; TSP1=1.04±0.061; PTX3+TSP1=0.968±0.076; E123=0.971±0.063; PTX3+E123=1.613±0.077. Number of dendrites examined: 103, 80, 88, 57, 51, 62 respectively; Kruskall-Wallis test, p<0.0001 followed by Dunn's test as indicated in figure; at least 3 independent experiments, data are presented as normalized mean values±SEM).


A ) Representative images of 14DIV neurons stained for surface AMPARs ( GluA , green ) , Bassoon ( blue ) and tubulin ( red ) in the different tested conditions . Arrowheads point to postsynaptic GluA clusters . Scale bar : 5µm . B ) Quantification of the surface synaptic AMPARs normalized to the total number of Bsn shows a decrease of the surface synaptic AMPAR clusters upon chronic application of the PTX3 blocking antibody to mixed cultures ( Ctr = 1 . 000 ± 0 . 063 , PTX3 block = 0 . 694 ± 0 . 069 , isotype Ab = 0 . 876 ± 0 . 077 . Number of fields examined : 34 , 23 , 19 respectively ; one - way ANOVA analysis of variance , p < 0 . 0001 followed by post hoc Tukey test as indicated in figure ; 3 independent experiments , data are presented as normalized mean values ± 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

A) Representative images of 14DIV neurons stained for surface AMPARs (GluA, green), Bassoon (blue) and tubulin (red) in the different tested conditions. Arrowheads point to postsynaptic GluA clusters. Scale bar: 5µm. B) Quantification of the surface synaptic AMPARs normalized to the total number of Bsn shows a decrease of the surface synaptic AMPAR clusters upon chronic application of the PTX3 blocking antibody to mixed cultures (Ctr=1.000±0.063, PTX3 block=0.694±0.069, isotype Ab=0.876±0.077. Number of fields examined: 34, 23, 19 respectively; one-way ANOVA analysis of variance, p<0.0001 followed by post hoc Tukey test as indicated in figure; 3 independent experiments, data are presented as normalized mean values ±SEM)


C ) Representative images of 14DIV WT and PTX3 KO neurons stained for surface AMPARs ( GluA , green ) , Bassoon ( blue ) and tubulin ( red ) . Arrowheads point to postsynaptic GluA clusters . Scale bar : 5µm . D ) Quantification of the surface synaptic AMPARs normalized to the total number of Bsn shows a reduction in PTX3 KO cultures with respect to WT ( WT = 1 ± 0 . 065 , KO = 0 . 771 ± 0 . 0 . 92 ; number of fields examined : 36 and 42 respectively ; Mann - Whitney test ; number of animals 5 WT and 6 PTX3 KO , data are presented as normalized mean values ± 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

C) Representative images of 14DIV WT and PTX3 KO neurons stained for surface AMPARs (GluA, green), Bassoon (blue) and tubulin (red). Arrowheads point to postsynaptic GluA clusters. Scale bar: 5µm. D) Quantification of the surface synaptic AMPARs normalized to the total number of Bsn shows a reduction in PTX3 KO cultures with respect to WT (WT=1±0.065, KO=0.771±0.0.92; number of fields examined: 36 and 42 respectively; Mann-Whitney test; number of animals 5 WT and 6 PTX3 KO, data are presented as normalized mean values±SEM)


E ) Examples of mEPSCs recorded in the indicated experimental conditions . F ) Quantitation of mEPSC inter - event interval showing a rescue of mEPSC frequency in PTX3 KO cultures treated with the N - terminal fragment of PTX3 ( WT = 1158 ± 34 . 67 ; WT + N - term = 979 . 1 ± 28 . 27 ; PTX3 KO = 1263 ± 38 . 99 ; PTX3 KO + Nterm = 1008 ± 30 . 14 . Number of neurons : WT , Ctr = 25 , WT + N - term = 29 ; PTX3 KO , Ctr = 24 ; PTX3 KO + N - term = 22 ; 3 independent experiments . Kruskall - Wallis test , p < 0 . 0001 followed by Dunn " s test as indicated in figure , data are presented as mean ± SEM ) . G ) Quantitation of mEPSC amplitude showing a rescue in PTX3 KO cultures treated with the N - terminal fragment of PTX3 ( pA , WT = 20 . 85 ± 0 . 249 WT + N - term = 22 . 37 ± 0 . 234 ; PTX3 KO = 20 . 31 ± 0 . 264 ; PTX3 KO + Nterm = 23 . 8 ± 0 . 316 . Number of neurons : WT , Ctr = 25 , WT + N - term = 29 ; PTX3 KO , Ctr = 24 ; PTX3 KO + N - term = 22 ; 3 independent experiments . Kruskall - Wallis test , p < 0 . 0001 followed by Dunn " s test as indicated in figure , data are presented

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Examples of mEPSCs recorded in the indicated experimental conditions. F) Quantitation of mEPSC inter-event interval showing a rescue of mEPSC frequency in PTX3 KO cultures treated with the N-terminal fragment of PTX3 (WT=1158±34.67; WT+N-term=979.1±28.27; PTX3 KO=1263±38.99; PTX3 KO+Nterm=1008±30.14. Number of neurons: WT, Ctr=25, WT+N-term= 29; PTX3 KO, Ctr= 24; PTX3 KO+N-term=22; 3 independent experiments. Kruskall-Wallis test, p<0.0001 followed by Dunn"s test as indicated in figure, data are presented as mean±SEM). G) Quantitation of mEPSC amplitude showing a rescue in PTX3 KO cultures treated with the N-terminal fragment of PTX3 (pA, WT=20.85±0.249 WT+N-term=22.37±0.234; PTX3 KO=20.31±0.264; PTX3 KO+Nterm=23.8±0.316. Number of neurons: WT, Ctr=25, WT+N-term= 29; PTX3 KO, Ctr= 24; PTX3 KO+N-term=22; 3 independent experiments. Kruskall-Wallis test, p<0.0001 followed by Dunn"s test as indicated in figure, data are presented as


H ) Representative images of 14DIV PTX3 KO neurons ( Ctr , + N terminal PTX3 , + C terminal PTX3 ) stained for surface AMPARs ( GluA , green ) , Bassoon ( blue ) and tubulin ( red ) . Arrowheads point to postsynaptic GluA clusters . Scale bar : 5µm . I ) Quantification of the surface synaptic AMPARs normalized to the total number of Bsn shows an increase in PTX3 KO treated with N terminal fragment of PTX3 but not with the C terminal fragment ( PTX3 KO = 1 . 000 ± 0 . 042 , PTX3 KO + C - term = 1 . 103 ± 0 . 070 , PTX3 KO + N - term = 1 . 428 ± 0 . 061 . Number of fields examined : 63 , 38 , 57 respectively ; Kruskall - Wallis test , p < 0 . 0001 followed by Dunn " s test ; 3 independent experiments , data are presented as normalized mean

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of 14DIV PTX3 KO neurons (Ctr, +N terminal PTX3, +C terminal PTX3) stained for surface AMPARs (GluA, green), Bassoon (blue) and tubulin (red). Arrowheads point to postsynaptic GluA clusters. Scale bar: 5µm. I) Quantification of the surface synaptic AMPARs normalized to the total number of Bsn shows an increase in PTX3 KO treated with N terminal fragment of PTX3 but not with the C terminal fragment (PTX3 KO=1.000±0.042, PTX3 KO+C-term=1.103±0.070, PTX3 KO+N-term=1.428±0.061. Number of fields examined: 63, 38, 57 respectively; Kruskall-Wallis test, p<0.0001 followed by Dunn"s test; 3 independent experiments, data are presented as normalized mean values


J - L ( J ) Examples of mEPSCs recordings in WT and PTX3 KO ( littermates ) hippocampal slices at P8 - 9 showing that frequency ( K ) and amplitude ( L ) are significantly decreased in KO neurons with respect to WT ( Hz : WT = 1 . 54 ± 0 . 136 ; PTX3 KO = 0 . 912 ± 0 . 083 ; Mann Whitney test . pA : WT = 16 . 57 ± 1 . 334 ; PTX3 KO = 12 . 55 ± 1 . 037 ; unpaired t - test . Data are presented as a distribution plus mean ± SEM . WT : 18 cells , 4 mice ; PTX3 KO : 20 cells , 5 mice )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-L (J) Examples of mEPSCs recordings in WT and PTX3 KO (littermates) hippocampal slices at P8-9 showing that frequency (K) and amplitude (L) are significantly decreased in KO neurons with respect to WT (Hz: WT=1.54±0.136; PTX3 KO=0.912±0.083; Mann Whitney test. pA: WT=16.57±1.334; PTX3 KO=12.55±1.037; unpaired t-test. Data are presented as a distribution plus mean±SEM. WT: 18 cells, 4 mice; PTX3 KO: 20 cells, 5 mice)


N ) Examples of mEPSCs recordings of P30 WT and PTX3 KO hippocampal slices . O ) Quantification of mEPSC frequency showing reduced frequency in PTX3 KO mice with respect to WT ( Hz : WT = 2 . 27 ± 0 . 115 ; PTX3 KO = 1 . 74 ± 0 . 136 . WT : 10 cells , 4 mice ; PTX3 KO : 11 cells , 5 mice . Unpaired t - test p = 0 . 008 . Data are presented as a distribution plus mean ± SEM ) . P , Q ( P ) No difference in the average amplitude is evident ( pA : WT = 11 . 95 ± 0 . 589 ; PTX3 KO = 10 . 69 ± 0 . 438 ; unpaired t - test p = 0 . 099 . Data are presented as a distribution plus mean ± SEM . WT : 10 cells , 4 mice ; PTX3 KO : 11 cells , 5 mice ) ; however the cumulative probability plot of amplitudes ( Q ) for mEPSCs in WT and PTX3 KO shows that there is a significant shift in the distribution by the Kolmogorov - Smirnov test ( p < 0 . 0001 , D = 0 . 112 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Examples of mEPSCs recordings of P30 WT and PTX3 KO hippocampal slices. O) Quantification of mEPSC frequency showing reduced frequency in PTX3 KO mice with respect to WT (Hz: WT=2.27±0.115; PTX3 KO=1.74±0.136. WT: 10 cells, 4 mice; PTX3 KO: 11 cells, 5 mice. Unpaired t-test p=0.008. Data are presented as a distribution plus mean±SEM). P, Q (P) No difference in the average amplitude is evident (pA: WT=11.95±0.589; PTX3 KO=10.69±0.438; unpaired t-test p=0.099. Data are presented as a distribution plus mean±SEM. WT: 10 cells, 4 mice; PTX3 KO: 11 cells, 5 mice); however the cumulative probability plot of amplitudes (Q) for mEPSCs in WT and PTX3 KO shows that there is a significant shift in the distribution by the Kolmogorov-Smirnov test (p <0.0001, D=0.112).


( B ) Epg5 protein and the wild - type copy of Epg5 are absent in Epg5 − / − mice . There is an intervening lane between Epg5 + / − and Epg5 − / − lanes in the Western blot .

O O O O O O O O O O O O O O O O O O O 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) Epg5 protein and the wild-type copy of Epg5 are absent in Epg5−/− mice. There is an intervening lane between Epg5+/− and Epg5−/− lanes in the Western blot.


( C ) Body weight curves of Epg5 + / − and Epg5 − / − males . Means ± SEM of 12 mice are shown .

O O O 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) Body weight curves of Epg5+/− and Epg5−/− males. Means ± SEM of 12 mice are shown.


( D ) Time spent on a rotarod . The horizontal lines indicate mean times .

(D) Time spent on a rotarod. The horizontal lines indicate mean times.


( E ) Epg5 − / − males show gradually worsening limb - clasping reflexes .

(E) Epg5−/− males show gradually worsening limb-clasping reflexes.


( F ) The hindquarters of Epg5 − / − mice are completely paralyzed at 10 mo of age .

(F) The hindquarters of Epg5−/− mice are completely paralyzed at 10 mo of age.


( G ) Spinal deformation in an Epg5 − / − male at 10 mo of age .

(G) Spinal deformation in an Epg5−/− male at 10 mo of age.


( H ) Epg5 − / − females gradually develop the limb - clasping phenotype .

(H) Epg5−/− females gradually develop the limb-clasping phenotype.


( A ) Nissl staining of the fifth layer of the cortex in 10 - mo - old Epg5 + / − and Epg5 − / − mice . ( B ) The number of pyramidal cells in the fifth layer per millimeters squared .

O O O O O O O O O O O O O O O O O O O O O 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) Nissl staining of the fifth layer of the cortex in 10-mo-old Epg5+/− and Epg5−/− mice. (B) The number of pyramidal cells in the fifth layer per millimeters squared.


( C and D ) H & amp ; amp ; E staining of the hippocampus shows reduced pyramidal cell numbers in Epg5 − / − mice . Means ± SEM of three mice are shown in B and D .

O O O O O O O O O O O O O O O O O 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) H&amp;E staining of the hippocampus shows reduced pyramidal cell numbers in Epg5−/− mice. Means ± SEM of three mice are shown in B and D.


( E ) The normally developed cerebellum in Epg5 − / − mice . ( F ) The thickness of the molecular layer in the cerebellum in mutant and control mice .

O O O O O O O O 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) The normally developed cerebellum in Epg5−/− mice. (F) The thickness of the molecular layer in the cerebellum in mutant and control mice.


( G and H ) Purkinje cells , stained by calbindin , are not reduced in number in Epg5 − / − mice . Means ± SEM of five mice are shown in F and H .

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

(G and H) Purkinje cells, stained by calbindin, are not reduced in number in Epg5−/− mice. Means ± SEM of five mice are shown in F and H.


( I ) Arrows indicate Pyknotic neurons in the anterior horn of Epg5 − / − mice .

(I) Arrows indicate Pyknotic neurons in the anterior horn of Epg5−/−mice.


( J ) The number of motor neurons in the spinal cord ( SC ) in Epg5 + / − and Epg5 − / − mice .

(J) The number of motor neurons in the spinal cord (SC) in Epg5+/− and Epg5−/−mice.


( K ) The numbers of Nissl - stained interneurons in the spinal cord are similar in control and Epg5 − / − mice . Means ± SEM of three mice are shown in J and K .

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(K) The numbers of Nissl-stained interneurons in the spinal cord are similar in control and Epg5−/− mice. Means ± SEM of three mice are shown in J and K.


( L ) Eosinophilic spheroids ( arrows ) accumulate in the thoracic spinal cord of Epg5 − / − mice .

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(L) Eosinophilic spheroids (arrows) accumulate in the thoracic spinal cord of Epg5−/− mice.


( M ) Eosinophilic spheroids are swollen degenerating axons ( arrow ) .

(M) Eosinophilic spheroids are swollen degenerating axons (arrow).


( N ) EM pictures showing degenerated axons ( arrows ) in the spinal cord of Epg5 − / − mice .

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(N) EM pictures showing degenerated axons (arrows) in the spinal cord of Epg5−/− mice.


( O and P ) GFAP staining in Epg5 + / − and Epg5 − / − mice . DCST , dorsal corticospinal tract .

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(O and P) GFAP staining in Epg5+/− and Epg5−/−mice. DCST, dorsal corticospinal tract.


( A ) Levels of LC3 and p62 in brain and spinal cord extracts from Epg5 + / − and Epg5 − / − mice . SC , spinal cord .

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(A) Levels of LC3 and p62 in brain and spinal cord extracts from Epg5+/− and Epg5−/− mice. SC, spinal cord.


( B ) Polyubiquitinated proteins in detergent - soluble ( S ) and - insoluble ( I ) fractions from brain and spinal cord homogenates of Epg5 + / − and Epg5 − / − mice .

(B) Polyubiquitinated proteins in detergent-soluble (S) and -insoluble (I) fractions from brain and spinal cord homogenates of Epg5+/− and Epg5−/− mice.


( C ) p62 aggregates and ubiquitin - positive aggregates are absent from Epg5 + / − mice but dramatically accumulate and are colocalized ( arrows ) in Epg5 − / − mice .

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(C) p62 aggregates and ubiquitin-positive aggregates are absent from Epg5+/− mice but dramatically accumulate and are colocalized (arrows) in Epg5−/− mice.


( D - F ) p62 aggregates are detected in neurons ( arrows ; D ) and oligodendrocytes ( arrows ; E ) but absent from astrocytes ( F ) in Epg5 − / − mice .

(D-F) p62 aggregates are detected in neurons (arrows; D) and oligodendrocytes (arrows; E) but absent from astrocytes (F) in Epg5−/− mice.


( G ) Cytoplasmic TDP - 43 aggregates accumulate in motor neurons in the anterior horn of spinal cord in Epg5 − / − mice . Arrows show cytoplasmic TDP - 43 aggregates . DSCT , dorsal corticospinal tract . ( H ) Percentages of cytoplasmic TDP - 43 aggregate - positive motor neurons in the fifth layer of cortex and anterior horn of the spinal cord in Epg5 + / − and Epg5 − / − mice . Means ± SEM of three mice are shown . LC , lateral column . Bars , 10 µm .

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(G) Cytoplasmic TDP-43 aggregates accumulate in motor neurons in the anterior horn of spinal cord in Epg5−/− mice. Arrows show cytoplasmic TDP-43 aggregates. DSCT, dorsal corticospinal tract. (H) Percentages of cytoplasmic TDP-43 aggregate-positive motor neurons in the fifth layer of cortex and anterior horn of the spinal cord in Epg5+/− and Epg5−/− mice. Means ± SEM of three mice are shown. LC, lateral column. Bars, 10 µm.


( A and B ) Electromyography of the gastrocnemius muscle from a 3 - mo - old Epg5 − / − mouse showing fibrillations ( A ) and positive sharp waves ( B ) . These defects were absent in control mice .

(A and B) Electromyography of the gastrocnemius muscle from a 3-mo-old Epg5−/− mouse showing fibrillations (A) and positive sharp waves (B). These defects were absent in control mice.


( C ) At 10 mo , Epg5 − / − mice showed high - amplitude and long - duration action potentials when conducting MUAP tests . The blue lines show the start and end of an action potential . Dur , duration ; Amp , amplitude .

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(C) At 10 mo, Epg5−/−mice showed high-amplitude and long-duration action potentials when conducting MUAP tests. The blue lines show the start and end of an action potential. Dur, duration; Amp, amplitude.


( D - F ) H & amp ; amp ; E staining of gastrocnemius muscles showed features of muscle degeneration in Epg5 − / − mice . The arrow in E indicates centrally nucleated fibers .

(D-F) H&amp;E staining of gastrocnemius muscles showed features of muscle degeneration in Epg5−/−mice. The arrow in E indicates centrally nucleated fibers.


( G ) The transcription levels of the atrophy - related genes were up - regulated in gastrocnemius muscles of Epg5 − / − mice .

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(G) The transcription levels of the atrophy-related genes were up-regulated in gastrocnemius muscles of Epg5−/−mice.


(A) Levels of LC3 and p62 in brain and spinal cord extracts from Epg5+/− and Epg5−/−mice. SC, spinal cord.


( J ) Levels of LC3 - II and p62 in muscle extracts from Epg5 + / − and Epg5 − / − mice .

(J) Levels of LC3-II and p62 in muscle extracts from Epg5+/− and Epg5−/− mice.


( K ) Percentage of p62 aggregate - positive myofibers in Epg5 + / − and Epg5 − / − mice . Means ± SEM of three mice are shown .

(K) Percentage of p62 aggregate-positive myofibers in Epg5+/− and Epg5−/− mice. Means ± SEM of three mice are shown.


( L ) Accumulation and colocalization of p62 and ubiquitin aggregates in the gastrocnemius muscles of Epg5 − / − mice but not controls . Bars : ( D - F and L ) 10 µm ; ( H and I ) 1 µm .

(L) Accumulation and colocalization of p62 and ubiquitin aggregates in the gastrocnemius muscles of Epg5−/− mice but not controls. Bars: (D-F and L) 10 µm; (H and I) 1 µm.


( A ) Number of LC3 puncta in Epg5 + / − ( control ) and Epg5 − / − MEFs .

(A) Number of LC3 puncta in Epg5+/− (control) and Epg5−/− MEFs.


( B ) Western blot showing levels of LC3 - I and LC3 - II in control and Epg5 − / − MEFs upon indicated treatment ( Starv . , starvation ; Rapa . , rapamycin ; Bafilo . , bafilomycin A1 ) .

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(B) Western blot showing levels of LC3-I and LC3-II in control and Epg5−/− MEFs upon indicated treatment (Starv., starvation; Rapa., rapamycin; Bafilo., bafilomycin A1).


( C ) Proteinase K ( ProK ) protection assay in different MEFs . PNS , postnuclear supernatant ; P , pellet fractions .

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(C) Proteinase K (ProK) protection assay in different MEFs. PNS, postnuclear supernatant; P, pellet fractions.


( D ) Colocalization ratio of LC3 and LAMP - 1 in control and Epg5 − / − MEFs . 50 cells were examined per time point in A and D .

(D) Colocalization ratio of LC3 and LAMP-1 in control and Epg5−/− MEFs. 50 cells were examined per time point in A and D.


( E ) Colocalization of LC3 puncta and lysosomes in control and Epg5 − / − MEFs 4 h after starvation .

(E) Colocalization of LC3 puncta and lysosomes in control and Epg5−/− MEFs 4 h after starvation.


( F ) Under nutrient repletion conditions , almost no autophagic elements are detected in Epg5 + / − MEFs . RER , rough ER ; M , mitochondrion ; N , nucleus . The arrowhead indicates a vacuole with a late residual body - like appearance ( aAV - III ) . ( G ) Under nutrient repletion conditions , Epg5 − / − MEFs accumulate a large number of autophagic vacuoles . Red arrow , a likely autophagosome ; red arrowheads , complex vacuoles of the aAV - I type ; white arrowheads , aAV - II

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(F) Under nutrient repletion conditions, almost no autophagic elements are detected in Epg5+/− MEFs. RER, rough ER; M, mitochondrion; N, nucleus. The arrowhead indicates a vacuole with a late residual body-like appearance (aAV-III). (G) Under nutrient repletion conditions, Epg5−/− MEFs accumulate a large number of autophagic vacuoles. Red arrow, a likely autophagosome; red arrowheads, complex vacuoles of the aAV-I type; white arrowheads, aAV-II vacuoles


( H and I ) High magnification of autophagic elements in unstarved Epg5 − / − MEFs . ( H ) The red arrow indicates a complex autophagic vacuole containing multilayered membrane structures ( white arrowheads ) and a lipid - like region ( L ) . ( I ) The red arrow indicates an autophagosome containing cytoplasm and ribosomes . The red arrowhead points to an autophagosome containing a mitochondrion and cytoplasmic material with membranous structures . The white arrow shows an aAV - III vacuole .

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(H and I) High magnification of autophagic elements in unstarved Epg5−/− MEFs. (H) The red arrow indicates a complex autophagic vacuole containing multilayered membrane structures (white arrowheads) and a lipid-like region (L). (I) The red arrow indicates an autophagosome containing cytoplasm and ribosomes. The red arrowhead points to an autophagosome containing a mitochondrion and cytoplasmic material with membranous structures. The white arrow shows an aAV-III vacuole.


( J ) Compared with control cells , Rab5 - labeled early endosomes are significantly enlarged in Epg5 siRNA - treated cells .

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(J) Compared with control cells, Rab5-labeled early endosomes are significantly enlarged in Epg5 siRNA-treated cells.


( K and L ) EGFR degradation in control and Epg5 knockdown cells .

(K and L) EGFR degradation in control and Epg5 knockdown cells.


( M and N ) Distribution of Alexa Fluor 488 - conjugated transferrin at different time points in control siRNA - and Epg5 siRNA - treated cells . Higher magnification views are shown in the insets . Error bars indicate SEMs .

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(M and N) Distribution of Alexa Fluor 488-conjugated transferrin at different time points in control siRNA- and Epg5 siRNA-treated cells. Higher magnification views are shown in the insets. Error bars indicate SEMs.


( B ) Representative H / E images of epidermis from control and iDKO mice at 15 DPI . ( C ) Quantitative analysis of epidermal cell rounding from ( B ) . n = 3 biological replicates per genotype .

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(B) Representative H/E images of epidermis from control and iDKO mice at 15 DPI. (C) Quantitative analysis of epidermal cell rounding from (B). n=3 biological replicates per genotype.


( E - F ) Representative images ( E ) and quantification ( F ) of whole - mount immunostaining of β - catenin . Control : n = 7 biological replicates ; iDKO : n = 5 biological replicates .

(E-F) Representative images (E) and quantification (F) of whole-mount immunostaining of β-catenin. Control: n=7 biological replicates; iDKO: n=5 biological replicates.


( L ) Representative immunofluorescent images of the toe pad for filaggrin ( Flg ) . K14 marks basal keratinocytes and DAPI marks nuclei . ( M ) Quantification of the average size of filaggrin + deposits at 52 DPI as shown in ( L ) . n = 3 biological replicates per genotype .

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(L) Representative immunofluorescent images of the toe pad for filaggrin (Flg). K14 marks basal keratinocytes and DAPI marks nuclei. (M) Quantification of the average size of filaggrin+ deposits at 52 DPI as shown in (L). n=3 biological replicates per genotype.


(H) Volcano plots at 25 or 52 DPI.


( J ) RT - qPCR of the indicated genes in epidermis at 25 ( n = 4 biological replicates per genotype ) or 52 ( control : n = 10 biological replicates ; iDKO : n = 6 biological replicates ) DPI .

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(J) RT-qPCR of the indicated genes in epidermis at 25 (n=4 biological replicates per genotype) or 52 (control: n=10 biological replicates; iDKO: n=6 biological replicates) DPI.


( D ) ChIP - seq tracks of Ovol1 , Ovol2 , and histone marks for the select loci . Boxed areas indicate Ovol1 / 2 - binding peaks , and red bars underneath indicate presence of Ovol1 / 2 sequence motifs .

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(D) ChIP-seq tracks of Ovol1, Ovol2, and histone marks for the select loci. Boxed areas indicate Ovol1/2-binding peaks, and red bars underneath indicate presence of Ovol1/2 sequence motifs.


( F - G ) Luminex measurements of the indicated cytokines in skin lysates ( F ; n = 7 biological replicates per genotype ) or serum ( G ; n = 8 biological replicates per genotype ) of control and iDKO mice at 4 - 6 MPI .

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(F-G) Luminex measurements of the indicated cytokines in skin lysates (F; n=7 biological replicates per genotype) or serum (G; n=8 biological replicates per genotype) of control and iDKO mice at 4-6 MPI.


( A ) Representative whole - mount immunofluorescent staining of LCs in mouse back skin at 52 DPI . ( B ) Quantification of primary dendrites in LCs as shown in ( A ) . Control : n = 5 biological replicates ; iDKO : n = 4 biological replicates .

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(A) Representative whole-mount immunofluorescent staining of LCs in mouse back skin at 52 DPI. (B) Quantification of primary dendrites in LCs as shown in (A). Control: n=5 biological replicates; iDKO: n=4 biological replicates.


( C - D ) Flow cytometry analysis of isolated epidermis at 52 DPI . Control : n = 8 biological replicates ; iDKO : n = 7 biological replicates . MFI , mean fluorescence intensity .

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(C-D) Flow cytometry analysis of isolated epidermis at 52 DPI. Control: n=8 biological replicates; iDKO: n=7 biological replicates. MFI, mean fluorescence intensity.


( J - M ) Flow cytometry analysis of LNs at 52 DPI . n = 4 pairs for ( J ) . Control : n = 10 biological replicates ; iDKO : n = 5 biological replicates for ( K - M ) .

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(J-M) Flow cytometry analysis of LNs at 52 DPI. n=4 pairs for (J). Control: n=10 biological replicates; iDKO: n=5 biological replicates for (K-M).


( N ) RT - qPCR of the indicated genes in LNs from 4 - 6 MPI . n = 4 biological replicates per genotype .

(N) RT-qPCR of the indicated genes in LNs from 4-6 MPI. n=4 biological replicates per genotype.


( O ) Luminex measurements of G - CSF in serum of control and iDKO mice at 4 - 6 MPI . Left , results obtained using remote service at Eve Technologies ( n = 3 biological replicates per genotype ) . Right , results obtained in - house using a custom panel ( n = 8 biological replicates per genotype ) .

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(O) Luminex measurements of G-CSF in serum of control and iDKO mice at 4-6 MPI. Left, results obtained using remote service at Eve Technologies (n=3 biological replicates per genotype). Right, results obtained in-house using a custom panel (n=8 biological replicates per genotype).


( P ) Luminex measurements of IL - 17 in serum of control and iDKO mice at 52 DPI . n = 5 biological replicates per genotype .

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(P) Luminex measurements of IL-17 in serum of control and iDKO mice at 52 DPI. n=5 biological replicates per genotype.


( F ) Representative H / E images of control and iDKO skin at the indicated times showing changes in dWAT . ( G ) Quantification of fat cell area in ( F ) . n = 4 biological replicates per genotype . For , data is represented as mean ± SD and statistical analysis was done with the Holm - Sidak method . NS , non - significant ; * , p ≤ 0 . 05 ; * * , p ≤ 0 . 005 ; * * * , p ≤ 0 . 0005 ; * * * * , p ≤ 0 . 00005 .

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(F) Representative H/E images of control and iDKO skin at the indicated times showing changes in dWAT. (G) Quantification of fat cell area in (F). n=4 biological replicates per genotype. For , data is represented as mean ± SD and statistical analysis was done with the Holm-Sidak method. NS, non-significant; *, p≤0.05; **, p≤0.005; ***, p≤0.0005; ****, p≤0.00005.


( H - L ) Results from metabolic cage analysis of 4 - 5 - MPI control and iDKO mice ( n = 5 biological replicates per genotype ) at RT ( white band ) or after CE ( blue band ) . Gray bands designate night . Shown are data on food intake ( H ) , movement ( I ) , body weight ( J ) , energy expenditure ( K ) , and RER ( L ) .

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(H-L) Results from metabolic cage analysis of 4-5-MPI control and iDKO mice (n=5 biological replicates per genotype) at RT (white band) or after CE (blue band). Gray bands designate night. Shown are data on food intake (H), movement (I), body weight (J), energy expenditure (K), and RER (L).


( C - D ) Representative H / E images ( C ) and quantitative analysis ( D ) of epidermal cell rounding , in control and iDKO mice at 4 MPI . n = 4 biological replicates for PBS - iDKO ; n = 5 biological replicates for all else .

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(C-D) Representative H/E images (C) and quantitative analysis (D) of epidermal cell rounding, in control and iDKO mice at 4 MPI. n=4 biological replicates for PBS-iDKO; n=5 biological replicates for all else.


( E ) Representative immunofluorescent images of back skin for Flg , K14 , and DAPI .

(E) Representative immunofluorescent images of back skin for Flg, K14, and DAPI.


( G ) Quantification of LN weight . Control - PBS : n = 10 biological replicates ; iDKO - PBS : n = 4 biological replicates ; Control - Dex : n = 9 biological replicates ; iDKO - Dex : n = 7 biological replicates . ( H - J ) Flow cytometry analysis of LNs at 4 MPI . Control - PBS : n = 8 biological replicates ; iDKO - PBS : n = 4 biological replicates ; Control - Dex : n = 5 biological replicates ; iDKO - Dex : n = 4 biological replicates for ( H , J ) and Control - PBS : n = 7 biological replicates ; iDKO - PBS : n = 4 biological replicates ; Control - Dex : n = 5 biological replicates ; iDKO - Dex : n = 3 biological replicates for ( I ) .

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(G) Quantification of LN weight. Control-PBS: n=10 biological replicates; iDKO-PBS: n=4 biological replicates; Control-Dex: n=9 biological replicates; iDKO-Dex: n=7 biological replicates. (H-J) Flow cytometry analysis of LNs at 4 MPI. Control-PBS: n=8 biological replicates; iDKO-PBS: n=4 biological replicates; Control-Dex: n=5 biological replicates; iDKO-Dex: n=4 biological replicates for (H,J) and Control-PBS: n=7 biological replicates; iDKO-PBS: n=4 biological replicates; Control-Dex: n=5 biological replicates; iDKO-Dex: n=3 biological replicates for (I).


( L ) Body weight over time represented as a percentage of the starting weight . Control - PBS : n = 13 biological replicates ; iDKO - PBS : n = 4 biological replicates ; Control - Dex : n = 9 biological replicates ; iDKO - Dex : n = 5 biological replicates .

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(L) Body weight over time represented as a percentage of the starting weight. Control-PBS: n=13 biological replicates; iDKO-PBS: n=4 biological replicates; Control-Dex: n=9 biological replicates; iDKO-Dex: n=5 biological replicates.


A Western blot analysis of B16F1 WT and two independent XIAPKO cell lines . Actin was used as a loading control .

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A Western blot analysis of B16F1 WT and two independent XIAPKO cell lines. Actin was used as a loading control.


B Subcutaneous melanoma tumour growth in BL / 6 WT mice ( n = 5 ) using B16F1 WT and XIAPKO ( clone1 and 2 ) .

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B Subcutaneous melanoma tumour growth in BL/6 WT mice (n=5) using B16F1 WT and XIAPKO (clone1 and 2).


C Representative H & E ( scale bar 50 µm ) and fluorescent images of cl - Caspase 3 staining in B16 WT and XIAPKO tumours . Nuclei were stained with DAPI ( blue ) . Scale bar 100 µm . Quantification thereof ( right panel ) . The average of cl - Caspase 3 positive cells from 5 randomly selected areas of the tumour ( 5 mice per genotype ) was represented . Dots represent individual mice ( n = 5 ) .

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C Representative H&E (scale bar 50 µm) and fluorescent images of cl-Caspase 3 staining in B16 WT and XIAPKO tumours. Nuclei were stained with DAPI (blue). Scale bar 100 µm. Quantification thereof (right panel). The average of cl-Caspase 3 positive cells from 5 randomly selected areas of the tumour (5 mice per genotype) was represented. Dots represent individual mice (n=5).


D qRT - PCR analysis ( PrimePCRTM Assay ) of different cyto - / chemokines in B16F1 WT cells plotted vs B16F1 - XIAPKO cells ( clone1 and 2 ) . Samples that are at least 2 - fold up - regulated are shown in red , samples that are at least 2 - fold down - regulated are shown in green .

D qRT-PCR analysis (PrimePCRTM Assay) of different cyto-/chemokines in B16F1 WT cells plotted vs B16F1-XIAPKO cells (clone1 and 2). Samples that are at least 2-fold up-regulated are shown in red, samples that are at least 2-fold down-regulated are shown in green.


E IL8 measurement in the supernatant of the indicated cell lines after 48 h transfection with siScr ( control ) or siXIAP and Western blot analysis of the corresponding cell lysates ( bottom ) .

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E IL8 measurement in the supernatant of the indicated cell lines after 48 h transfection with siScr (control) or siXIAP and Western blot analysis of the corresponding cell lysates (bottom).


F IL8 measurement in the supernatant of the indicated cell lines after 24 h of seeding the cells .

F IL8 measurement in the supernatant of the indicated cell lines after 24 h of seeding the cells.


G qRT - PCR analysis ( PrimePCRTM Assay ) of different cyto - / chemokines in HEK293T cells overexpressing myc - XIAP plotted vs HEK293T cells overexpressing myc - XIAPΔRING . Samples that are at least 2 - fold up - regulated are shown in red , samples that are at least 2 - fold down - regulated are shown in green .

G qRT-PCR analysis (PrimePCRTM Assay) of different cyto-/chemokines in HEK293T cells overexpressing myc-XIAP plotted vs HEK293T cells overexpressing myc-XIAPΔRING. Samples that are at least 2-fold up-regulated are shown in red, samples that are at least 2-fold down-regulated are shown in green.


H Human cytokines array analysis of HEK293T cells transfected with myc - XIAP or myc - XIAPΔRING . Medium was changed after 16 h and the supernatant was collected after 48 h . Pre - spotted nitrocellulose membranes were incubated with the supernatants overnight at 4 ° C .

H Human cytokines array analysis of HEK293T cells transfected with myc-XIAP or myc-XIAPΔRING. Medium was changed after 16 h and the supernatant was collected after 48 h. Pre-spotted nitrocellulose membranes were incubated with the supernatants overnight at 4°C.


A Western blot analysis of cell lysates of BLM and BLM - XIAPKO cells ( input ) and XIAP - IP . Data are representative of two experiments .

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A Western blot analysis of cell lysates of BLM and BLM-XIAPKO cells (input) and XIAP-IP. Data are representative of two experiments.


B Confocal microscopic analysis of transfected HCT116 cells with GFP - XIAP for 16 h and stained for TAB1 or RIPK2 ( red ) . Nuclei were stained with DAPI ( blue ) .

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B Confocal microscopic analysis of transfected HCT116 cells with GFP-XIAP for 16 h and stained for TAB1 or RIPK2 (red). Nuclei were stained with DAPI (blue).


C Cell - free ubiquitylation assay of recombinant GST - RIPK2 by Flag - XIAP after addition of respective E1 / E2 and recombinant ubiquitin . Poly - ubiquitylated RIPK2 and XIAP were detected by Western blotting .

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C Cell-free ubiquitylation assay of recombinant GST-RIPK2 by Flag-XIAP after addition of respective E1/E2 and recombinant ubiquitin. Poly-ubiquitylated RIPK2 and XIAP were detected by Western blotting.


D Western blot analysis of cell lysates of BLM , BLM - XIAPKO , SK - Mel28 , SK - Mel28 - XIAPKO , B16 , and B16 - XIAPKO cells ( input ) and RIPK2 - IP . Poly - ubiquitin is detected using anti - ubiquitin antibody .

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D Western blot analysis of cell lysates of BLM, BLM-XIAPKO, SK-Mel28, SK-Mel28-XIAPKO, B16, and B16-XIAPKO cells (input) and RIPK2-IP. Poly-ubiquitin is detected using anti-ubiquitin antibody.


E IL8 measurement in the supernatant of transfected BLM ( left panel ) or SK - Mel28 ( right panel ) cells with siScr ( control ) , siXIAP , siTAB1 or siRIPK2 after 48 h . Western blot analysis of the respective cell lysates of BLM cells ( left bottom ) or SK - Mel28 cells ( right bottom ) transfected with the indicated siRNA .

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E IL8 measurement in the supernatant of transfected BLM (left panel) or SK-Mel28 (right panel) cells with siScr (control), siXIAP, siTAB1 or siRIPK2 after 48 h. Western blot analysis of the respective cell lysates of BLM cells (left bottom) or SK-Mel28 cells (right bottom) transfected with the indicated siRNA.


F IL8 measurement in the supernatant of BLM or SK - Mel28 treated with 2 µM d89 ( BIR2 antagonist ) or DMSO as a negative control for 24 h .

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F IL8 measurement in the supernatant of BLM or SK-Mel28 treated with 2 µM d89 (BIR2 antagonist) or DMSO as a negative control for 24 h.


G IL8 measurement in the supernatant of BLM or SK - Mel28 treated with 20 µM birinapant ( Bir ) , 2 µM BV6 ( both BIR3 antagonist ) or DMSO as a negative control for 24 h .

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G IL8 measurement in the supernatant of BLM or SK-Mel28 treated with 20 µM birinapant (Bir), 2 µM BV6 (both BIR3 antagonist) or DMSO as a negative control for 24 h.


A Intensities of CD177 and XIAP immunostainings in tumour sections from 55 patients were visually scored . The numbers of melanomas with different intensities were plotted into the table as a percentage of the total . Specific staining intensity for XIAP and CD177 , corresponding to relative amounts of infiltrated neutrophils , was arbitrarily set as the following : - , not expressed ; + , low ; + + , moderate ; and + + + , strong expression . Representative IHC pictures ( right panel ) .

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A Intensities of CD177 and XIAP immunostainings in tumour sections from 55 patients were visually scored. The numbers of melanomas with different intensities were plotted into the table as a percentage of the total. Specific staining intensity for XIAP and CD177, corresponding to relative amounts of infiltrated neutrophils, was arbitrarily set as the following: -, not expressed; +, low; ++, moderate; and +++, strong expression. Representative IHC pictures (right panel).


B Representative IF ( neutrophils staining ) in B16F1 WT , XIAPKO , TAB1KO or RIPK2KO tumours ( Scale bar 50 µm ) and quantification of Ly6G + cells . The sum of Ly6G - positive cells from 5 randomly selected areas of the tumour ( 5 mice per genotype ) was represented . Dots represent individual mice ( n = 5 ) . C B16F1 WT , XIAPKO ( clone 2 ) subcutaneous melanoma tumour growth in BL / 6 WT mice ( n = 5 ) . Mice were intra - peritoneal injected with IgG antibody as a negative control or Ly6G antibody 1 day prior to the subcutaneous injection of the B16F1 WT or XIAPKO cells .

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B Representative IF (neutrophils staining) in B16F1 WT, XIAPKO, TAB1KO or RIPK2KO tumours (Scale bar 50 µm) and quantification of Ly6G+ cells. The sum of Ly6G-positive cells from 5 randomly selected areas of the tumour (5 mice per genotype) was represented. Dots represent individual mice (n=5). C B16F1 WT, XIAPKO (clone 2) subcutaneous melanoma tumour growth in BL/6 WT mice (n=5). Mice were intra-peritoneal injected with IgG antibody as a negative control or Ly6G antibody 1 day prior to the subcutaneous injection of the B16F1 WT or XIAPKO cells.


D Representative IF ( neutrophils ) scale bar 50 µm and quantification of Ly6G + cells in tumours from C . The sum of Ly6G - positive cells from 5 randomly selected areas of the tumour ( 4 mice per genotype ) was represented . Dots represent individual mice ( n = 4 ) .

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D Representative IF (neutrophils) scale bar 50 µm and quantification of Ly6G+ cells in tumours from C. The sum of Ly6G-positive cells from 5 randomly selected areas of the tumour (4 mice per genotype) was represented. Dots represent individual mice (n=4).


C Maximal cytochrome c release for all DX / REL pairs with ≥ 3 biological replicates ) ; box - whisker plots summarize data ( box 25 % - 75 % ; belt = median ; dot = mean ; whiskers = minimum and maximum values ) . Statistical analyses were performed using an unpaired two - sided Student ' s t - test , with significance p < 0 . 05 ( trend p < 0 . 10 ) . Cyto c , cytochrome

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C Maximal cytochrome c release for all DX/REL pairs with ≥3 biological replicates); box-whisker plots summarize data (box 25%-75%; belt=median; dot=mean; whiskers=minimum and maximum values). Statistical analyses were performed using an unpaired two-sided Student's t-test, with significance p<0.05 (trend p<0.10). Cyto c, cytochrome c


D Bak oligomerization in response to escalating tBid concentration for DX / REL pairs . Relative mitochondrial protein loading per lane is assessed by densitometry , showing ration of loading control in REL lane with patient - matched DX lane at same tBid exposure ( no REL lane is underloaded compared to DX lane ) .

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D Bak oligomerization in response to escalating tBid concentration for DX/REL pairs. Relative mitochondrial protein loading per lane is assessed by densitometry, showing ration of loading control in REL lane with patient-matched DX lane at same tBid exposure (no REL lane is underloaded compared to DX lane).


A In vitro viability curves after 72hr exposure to mafosfamide or doxorubicin . Results are shown for three DX / REL neuroblastoma cell line pairs , two with attenuated cytochrome c release ( CHLA15 / CHLA20 and SKNBE1 / SKNBE2C ) and one without ( CHLA122 / CHLA136 ) . Data information : data points are mean and SD from triplicate wells , experiments are representative of at least three biological replicates dotted - line represents 50 % viability .

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A In vitro viability curves after 72hr exposure to mafosfamide or doxorubicin. Results are shown for three DX/REL neuroblastoma cell line pairs, two with attenuated cytochrome c release (CHLA15/CHLA20 and SKNBE1/SKNBE2C) and one without (CHLA122/CHLA136). Data information: data points are mean and SD from triplicate wells, experiments are representative of at least three biological replicates dotted-line represents 50% viability.


C , In vitro viability for CHLA15 ( DX ) and CHLA20 ( REL ) following ionizing radiation at 7 days Data information : data points are mean and SD from triplicate wells , experiments are representative of at least three biological replicates , dotted - line represents 50 % viability .

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C, In vitro viability for CHLA15 (DX) and CHLA20 (REL) following ionizing radiation at 7 days Data information: data points are mean and SD from triplicate wells, experiments are representative of at least three biological replicates, dotted-line represents 50% viability.


F Cytochrome c release from mitochondria after exposure to tBid or BimBH3 peptide for parental NB1643 and SY5Y cells , in comparison to cells cultured in escalating concentrations of crizotinib until resistant ( NB1643 - ALKR and SY5Y - ALKR ) . Data information : data points are mean of duplicate wells ( SD < 0 . 05 at all points ) in a representative experiment from at least two biological replicates .

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F Cytochrome c release from mitochondria after exposure to tBid or BimBH3 peptide for parental NB1643 and SY5Y cells, in comparison to cells cultured in escalating concentrations of crizotinib until resistant (NB1643-ALKR and SY5Y-ALKR). Data information: data points are mean of duplicate wells (SD<0.05 at all points) in a representative experiment from at least two biological replicates.


A , B , C Transmission electron microscopy image analysis was used to quantify mitochondrial size , circularity and roundness in DX / REL neuroblastoma pairs ( mean + / - SD shown ) . Data information : , n = 137 - 241 mitochondria per cell line statistical analyses were performed using a two - tailed Mann - Whitney U test , with significance p < 0 . 05 ( trend p < 0 . 10 ) .

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A, B, C Transmission electron microscopy image analysis was used to quantify mitochondrial size, circularity and roundness in DX/REL neuroblastoma pairs (mean +/- SD shown). Data information: , n=137-241 mitochondria per cell line statistical analyses were performed using a two-tailed Mann-Whitney U test, with significance p<0.05 (trend p<0.10).


H In vitro viability of CHLA15 - ctrl , CHLA15 - shMFN2 , CHLA15 - shPACS2 , and CHLA20 cells following 72hr exposure to ABT - 737 ; dotted - line represents 50 % viability . Data information : data points are mean and SD from triplicate wells , experiments are representative of at least two biological replicates .

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H In vitro viability of CHLA15-ctrl, CHLA15-shMFN2, CHLA15-shPACS2, and CHLA20 cells following 72hr exposure to ABT-737; dotted-line represents 50% viability. Data information: data points are mean and SD from triplicate wells, experiments are representative of at least two biological replicates.


I Mitochondrial cytochrome c release in response to tBid and BimBH3 peptide in CHLA15 - Ctrl , CHLA15 - shMFN2 and CHLA15 - shPACS2 cells . Data information : , data points are mean of duplicate wells ( SD < 0 . 05 at all points ) in a representative experiment from at least two biological

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I Mitochondrial cytochrome c release in response to tBid and BimBH3 peptide in CHLA15-Ctrl, CHLA15-shMFN2 and CHLA15-shPACS2 cells. Data information: , data points are mean of duplicate wells (SD<0.05 at all points) in a representative experiment from at least two biological replicates


A , B Cytosolic ( black - tracing ) and mitochondrial ( colored - tracing ) Ca2 + concentration measured using fluorescent reporters in the CHLA15 ( n = 29 cells ) / CHLA20 ( n = 65 cells ) and SKNBE1 ( n = 37 cells ) / SKNBE2C ( n = 52 cells ) pairs . ER Ca2 + release was induced by 100μM carbachol , an IP3R - agonist ; time added indicated by arrow ; tracings are mean + / - SD error bars . Coupling time ( time between achieving 50 % of maximal cytosolic and mitochondrial concentrations ) is an index of ER - mitochondrial proximity and transfer efficiency ; plotted below . Data information : data is derived from at least 3 separate cell transfections ( biological replicates ) . statistical analyses were performed using an unpaired two - sided Student ' s t - test , with significance p < 0 . 05 .

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A, B Cytosolic (black-tracing) and mitochondrial (colored-tracing) Ca2+ concentration measured using fluorescent reporters in the CHLA15 (n=29 cells)/CHLA20 (n=65 cells) and SKNBE1 (n=37 cells)/SKNBE2C (n=52 cells) pairs. ER Ca2+ release was induced by 100μM carbachol, an IP3R-agonist; time added indicated by arrow; tracings are mean +/- SD error bars. Coupling time (time between achieving 50% of maximal cytosolic and mitochondrial concentrations) is an index of ER-mitochondrial proximity and transfer efficiency; plotted below. Data information: data is derived from at least 3 separate cell transfections (biological replicates). statistical analyses were performed using an unpaired two-sided Student's t-test, with significance p<0.05.


F Cytochrome c release in response to tBid and BimBH3 assessed . Data information : data points are mean of duplicate wells ( SD < 0 . 05 at all points ) in a representative experiment from at least two biological replicates .

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F Cytochrome c release in response to tBid and BimBH3 assessed. Data information: data points are mean of duplicate wells (SD<0.05 at all points) in a representative experiment from at least two biological replicates.


A , B , C Concentration of ceramide species as measured by LC / MS from DX and REL pair whole cell pellets . Data information : show mean of 3 - 4 biological replicates plotted ; 3 technical replicates

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A, B, C Concentration of ceramide species as measured by LC/MS from DX and REL pair whole cell pellets. Data information: show mean of 3-4 biological replicates plotted; 3 technical replicates each


D , E Cumulative ceramides and sphingomyelins ( of C32 - C36 chain length ) for DX / REL pairs . Data information : show mean of 3 - 4 biological replicates plotted ; 3 technical replicates each error bars are + / - SD .

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D, E Cumulative ceramides and sphingomyelins (of C32-C36 chain length) for DX/REL pairs. Data information: show mean of 3-4 biological replicates plotted; 3 technical replicates each error bars are +/- SD.


H Summary data for CHLA15 / CHLA20 and SKNBE1 / SKNBE2C showing relative cytochrome c release when pre - incubated with GW4869 to inhibit ceramide generation , compared with untreated cells . Data information : All data points from two ( CHLA15 ) or three ( SKNBE1 ) biological replicates are depicted in H . Statistical analyses were performed using an unpaired two - sided Student ' s t - test , with significance p < 0 . 05 ( trend , p < 0 . 10 ) .

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H Summary data for CHLA15/CHLA20 and SKNBE1/SKNBE2C showing relative cytochrome c release when pre-incubated with GW4869 to inhibit ceramide generation, compared with untreated cells. Data information: All data points from two (CHLA15) or three (SKNBE1) biological replicates are depicted in H. Statistical analyses were performed using an unpaired two-sided Student's t-test, with significance p<0.05 (trend, p<0.10).


( B , C ) Body weight of the WT and L - HRD KO mice at the age of 12 weeks . ( n = 5 for each group ) Data information : The data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t test .

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(B, C) Body weight of the WT and L-HRD KO mice at the age of 12 weeks. (n=5 for each group) Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( D ) Body heights and tibia lengths of the WT and L - HRD KO mice . ( n = 5 for each group ) Data information : The data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t test .

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(D) Body heights and tibia lengths of the WT and L-HRD KO mice. (n=5 for each group) Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( E ) Phosphorylation of STAT5 in WT and HRD1 LKO liver .

(E) Phosphorylation of STAT5 in WT and HRD1 LKO liver.


( E ) Hepatic growth hormone signaling target genes mRNA levels in the WT and L - HRD1 KO mice . ( n = 5 for each group ) . Data information : The data are representative of three independent experiments ( mean ± s . d . ) .

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(E) Hepatic growth hormone signaling target genes mRNA levels in the WT and L-HRD1 KO mice. (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.).


Hepatic growth hormone signaling target genes mRNA levels in the WT and L - HRD1 KO mice . ( n = 5 for each group ) . Data information : The data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t test .

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Hepatic growth hormone signaling target genes mRNA levels in the WT and L-HRD1 KO mice. (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( A ) Volcano plot of differential genes between WT and HRD1 LKO livers .

(A) Volcano plot of differential genes between WT and HRD1 LKO livers.


( B ) Hepatic Fgf21 mRNA in the WT and L - HRD1 KO mice . ( n = 5 for each group ) . Data information : The data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t test .

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(B) Hepatic Fgf21 mRNA in the WT and L-HRD1 KO mice. (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( C ) Serum FGF21 protein levels in the WT and L - HRD1 KO mice . ( n = 5 for each group ) . Data information : The data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t test .

(C) Serum FGF21 protein levels in the WT and L-HRD1 KO mice. (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( D ) Correlation analysis of the gene expression ( Red : Upregulated genes and Blue : Downrelated genes ) between HRD1 LKO mice and FGF21 transgenic mice .

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(D) Correlation analysis of the gene expression (Red: Upregulated genes and Blue: Downrelated genes) between HRD1 LKO mice and FGF21 transgenic mice.


( E ) Percentage of mated with proven stud males and age at onset of puberty in the female WT and L - HRD KO mice . ( n = 10 for each group ) Data information : The data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t test .

(E) Percentage of mated with proven stud males and age at onset of puberty in the female WT and L-HRD KO mice. (n=10 for each group) Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( F ) Examples of ovarian histology from WT and HRD1 LKO mice .

(F) Examples of ovarian histology from WT and HRD1 LKO mice.


( G ) Representative examples of estrus cycles in WT and L - HRD1 KO mice as determined by vaginal cytology .

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(G) Representative examples of estrus cycles in WT and L-HRD1 KO mice as determined by vaginal cytology.


( H ) Percentage of feeding , water intake and activity in the light time . ( n = 8 for each group ) . Data information : The data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t test .

(H) Percentage of feeding, water intake and activity in the light time. (n=8 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( I ) H & E stain of white adipose tissue WT and HRD1 LKO mice 14 weeks after HFD feeding .

(I) H&E stain of white adipose tissue WT and HRD1 LKO mice 14 weeks after HFD feeding.


( J ) Ucp1 and Dio2 mRNA levels of adipose tissue from ( I ) ( n = 5 for each group ) . Data information : The data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t test .

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(J) Ucp1 and Dio2 mRNA levels of adipose tissue from (I) (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( B ) Volcano plot of differential proteins between WT and HRD1 LKO livers .

(B) Volcano plot of differential proteins between WT and HRD1 LKO livers.


( F ) Protein levels of IRE1α , NRF1 , RXRβ , NOTCH1 , SIRT3 and GAPDH in the liver of WT and HRD1 LKO mice under the refed condition . ( n = 6 for each group ) .

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(F) Protein levels of IRE1α, NRF1, RXRβ, NOTCH1, SIRT3 and GAPDH in the liver of WT and HRD1 LKO mice under the refed condition. (n=6 for each group).


Hepatic CREBH protein levels in the WT and L - HRD1 KO mice . ( n = 6 for each group ) .

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Hepatic CREBH protein levels in the WT and L-HRD1 KO mice. (n=6 for each group).


Hepatic CREBH mRNA ( B ) levels in the WT and L - HRD1 KO mice . ( n = 6 for each group ) . Data information : The data are representative of three independent experiments ( mean ± s . d . ) .

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Hepatic CREBH mRNA (B) levels in the WT and L-HRD1 KO mice. (n=6 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.).