E TNP - KLH specific IgG concentration in sera of mice from ( B ) collected before ( day 35 ; pre ) and after ( day 49 ; post ) the first boost ( n = 13 NC , 19 CPA 300 , 4 CPA 200 , 5 ALS , 6 ANTI - CD4 ; three independent experiments ) and at day 81 ( pre ) and 89 ( post ) for the second boost ( n = 4 NC , 4 CPA 300 , 4 CPA 200 , 5 ALS , 6 ANTI - CD4 ) . Sera from vaccinated WT ( n = 5 ) and Cd40lg - / - ( n = 5 ) mice were used as positive and negative controls , respectively . For early challenge data , comparisons were performed with an LME model , accounting for multiple experiments , followed by appropriate post - hoc analysis ( see Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to the overall difference among groups ( * * * p = 0 . 0002 and * * * * p < 0 . 0001 in all comparisons ) . CPA200 was not included in the analysis because n = 4 . WT group , not indicated in the figure , is significantly different from all groups ( p < 0 . 0001 in all comparisons ) . Of note , regarding the comparisons between time - points , the groups CPA 300 and WT show a significant increase between pre and post values ( p < 0 . 0001 for both ) , while all other groups show a significant decrease between pre and post values ( p < 0 . 0001 for all except p = 0 . 0022 for ANTI - CD4 ) . Median ± IQR .

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

E TNP-KLH specific IgG concentration in sera of mice from (B) collected before (day 35; pre) and after (day 49; post) the first boost (n=13 NC, 19 CPA 300, 4 CPA 200, 5 ALS, 6 ANTI-CD4; three independent experiments) and at day 81 (pre) and 89 (post) for the second boost (n=4 NC, 4 CPA 300, 4 CPA 200, 5 ALS, 6 ANTI-CD4). Sera from vaccinated WT (n=5) and Cd40lg-/- (n=5) mice were used as positive and negative controls, respectively. For early challenge data, comparisons were performed with an LME model, accounting for multiple experiments, followed by appropriate post-hoc analysis (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to the overall difference among groups (***p=0.0002 and ****p<0.0001 in all comparisons). CPA200 was not included in the analysis because n=4. WT group, not indicated in the figure, is significantly different from all groups (p<0.0001 in all comparisons). Of note, regarding the comparisons between time-points, the groups CPA 300 and WT show a significant increase between pre and post values (p<0.0001 for both), while all other groups show a significant decrease between pre and post values (p<0.0001 for all except p=0.0022 for ANTI-CD4). Median ± IQR.


F Total counts of recipient CD3 + cells gated within CD45 . 2 + cells after transfer of primed donor T cells in PB of mice treated with 300 mg / kg CPA ( n = 11 ) , 200 mg / kg CPA ( n = 11 ) or NC ( n = 6 ) . Longitudinal comparisons were performed by LME model followed by an appropriate post - hoc analysis ( Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to day 4 ( * * * * p < 0 . 0001 in all comparisons ) . Mean ± SEM .

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F Total counts of recipient CD3+ cells gated within CD45.2+ cells after transfer of primed donor T cells in PB of mice treated with 300 mg/kg CPA (n=11), 200 mg/kg CPA (n=11) or NC (n=6). Longitudinal comparisons were performed by LME model followed by an appropriate post-hoc analysis (Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to day 4 (****p<0.0001 in all comparisons). Mean ± SEM.


G Total counts of engrafted donor CD45 . 1 + cells in PB of mice from ( F ) . Longitudinal comparisons were performed with an LME model followed by an appropriate post - hoc analysis ( Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to day 25 . At day 215 a significant difference was observed between CPA 300 vs NC and CPA 300 vs CPA 200 ( * * * * p < 0 . 0001 for both ) . Mean ± SEM .

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G Total counts of engrafted donor CD45.1+ cells in PB of mice from (F). Longitudinal comparisons were performed with an LME model followed by an appropriate post-hoc analysis (Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to day 25. At day 215 a significant difference was observed between CPA 300 vs NC and CPA 300 vs CPA 200 (****p<0.0001 for both). Mean ± SEM.


H TNP - KLH specific IgG concentration in sera of mice from ( F ) collected before ( day 36 , pre ) and after ( day 50 , post ) the first boost ( n = 6 NC , 11 CPA 300 , 11 CPA 200 ) and at day 212 ( pre ) and 219 ( post ) for the second boost ( n = 6 NC , 11 CPA 300 , 9 CPA 200 ) . Sera from vaccinated WT ( n = 11 ) and Cd40lg - / - ( n = 5 ) mice were used as positive and negative controls , respectively . Comparisons were performed with an LME model followed by an appropriate post - hoc analysis , separately for each challenge data ( Appendix Supplementary Statistical Methods ) . Reported statistical comparisons refer to the overall difference among groups , for early challenge data ( * p = 0 . 0282 and * * p = 0 . 0037 ) , while to differences between time - points within each group , for late challenge data ( * * p = 0 . 0018 and * * * * p < 0 . 0001 in both comparisons ) . Of note , in the late challenge data , no significant overall differences were observed among the groups . Median ± IQR .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 TNP-KLH specific IgG concentration in sera of mice from (F) collected before (day 36, pre) and after (day 50, post) the first boost (n=6 NC, 11 CPA 300, 11 CPA 200) and at day 212 (pre) and 219 (post) for the second boost (n=6 NC, 11 CPA 300, 9 CPA 200). Sera from vaccinated WT (n=11) and Cd40lg-/- (n=5) mice were used as positive and negative controls, respectively. Comparisons were performed with an LME model followed by an appropriate post-hoc analysis, separately for each challenge data (Appendix Supplementary Statistical Methods). Reported statistical comparisons refer to the overall difference among groups, for early challenge data (*p=0.0282 and **p=0.0037), while to differences between time-points within each group, for late challenge data (**p=0.0018 and ****p<0.0001 in both comparisons). Of note, in the late challenge data, no significant overall differences were observed among the groups. Median ± IQR.


B Total counts of WT donor CD4 + T cells in mice from ( A ) ( n = 13 100 % WT , 8 25 % WT , 14 10 % WT , 6 1 % , 14 0 % WT ) . Two representative experiments shown out of 3 . Mean ± SEM .

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B Total counts of WT donor CD4+ T cells in mice from (A) (n=13 100% WT, 8 25% WT, 14 10% WT, 6 1%, 14 0% WT). Two representative experiments shown out of 3. Mean ± SEM.


C TNP - KLH specific IgG concentration in sera of mice from ( A ) collected seven days before ( pre ) and after ( post ) TNP - KLH vaccination ( n = 13 100 % WT , 13 25 % WT , 14 10 % WT , 6 1 % WT , 14 0 % WT ) . Three independent experiments . Comparisons were performed with an LME model , accounting for multiple experiments , followed by an appropriate post - hoc analysis ( Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to the overall difference among groups ( * * * * p < 0 . 0001 in all comparisons ) . Median ± IQR .

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C TNP-KLH specific IgG concentration in sera of mice from (A) collected seven days before (pre) and after (post) TNP-KLH vaccination (n=13 100% WT, 13 25% WT, 14 10% WT, 6 1% WT, 14 0% WT). Three independent experiments. Comparisons were performed with an LME model, accounting for multiple experiments, followed by an appropriate post-hoc analysis (Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to the overall difference among groups (****p<0.0001 in all comparisons). Median ± IQR.


D Percentage of PNA + GL7 + GC B cells within the spleen of mice from ( A ) ( n = 16 100 % WT , 6 25 % WT , 16 10 % WT , 11 1 % WT , 18 0 % WT ) . Kruskal - Wallis test followed by post - hoc analysis with Dunn ' s test . P - values were adjusted with Bonferroni ' s correction to account for multiple comparisons ( * p = 0 . 0385 for 25 % WT vs 1 % WT , * p = 0 . 0129 for 100 % WT vs 1 % WT , * * * p = 0 . 0001 and * * * * p < 0 . 0001 in both comparisons ) . Median ± IQR .

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D Percentage of PNA+GL7+ GC B cells within the spleen of mice from (A) (n=16 100% WT, 6 25% WT, 16 10% WT, 11 1% WT, 18 0% WT). Kruskal-Wallis test followed by post-hoc analysis with Dunn's test. P-values were adjusted with Bonferroni's correction to account for multiple comparisons (*p=0.0385 for 25% WT vs 1% WT, *p=0.0129 for 100% WT vs 1% WT, ***p=0.0001 and ****p<0.0001 in both comparisons). Median ± IQR.


E Quantitation of P . murina rRNA in lung homogenate of mice from ( A ) transplanted with different ratios of WT HSPC cells ( n = 8 100 % WT , 7 25 % WT , 7 10 % WT , 8 0 % WT ) and infected with the pathogen . Results are expressed in P . murina / HPRT RNA copies . Kruskal - Wallis test followed by post - hoc analysis with Dunn ' s test . P - values were adjusted with Bonferroni ' s correction to account for multiple comparisons . Only the groups 100 % WT and 0 % WT resulted to be significantly different ( * * p = 0 . 0028 ) . Median ± IQR .

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E Quantitation of P. murina rRNA in lung homogenate of mice from (A) transplanted with different ratios of WT HSPC cells (n=8 100% WT, 7 25% WT, 7 10% WT, 8 0% WT) and infected with the pathogen. Results are expressed in P.murina/HPRT RNA copies. Kruskal-Wallis test followed by post-hoc analysis with Dunn's test. P-values were adjusted with Bonferroni's correction to account for multiple comparisons. Only the groups 100% WT and 0% WT resulted to be significantly different (**p=0.0028). Median ± IQR.


F Lung weight of mice from ( E ) . Kruskal - Wallis test followed by post - hoc analysis with Dunn ' s test . P - values were adjusted with Bonferroni ' s correction to account for multiple comparisons . Only the groups 100 % WT and 0 % WT resulted to be significantly different ( * p = 0 . 0254 ) . Median ± IQR .

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F Lung weight of mice from (E). Kruskal-Wallis test followed by post-hoc analysis with Dunn's test. P-values were adjusted with Bonferroni's correction to account for multiple comparisons. Only the groups 100% WT and 0% WT resulted to be significantly different (*p=0.0254). Median ± IQR.


G Left : Representative pictures of lung tissue sections stained immunohistochemically with a rabbit primary antibody ( upper left : negative sample ; lower left : positive sample ) . Brown areas represent intra - alveolar aggregates of P . murina organisms . Right : Representative pictures of P . murina organisms detected by immunofluorescence in lungs homogenate ( upper right : negative sample ; lower right : positive sample ) .

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G Left: Representative pictures of lung tissue sections stained immunohistochemically with a rabbit primary antibody (upper left: negative sample; lower left: positive sample). Brown areas represent intra-alveolar aggregates of P. murina organisms. Right: Representative pictures of P. murina organisms detected by immunofluorescence in lungs homogenate (upper right: negative sample; lower right: positive sample).


H Quantitation of P . Murina rRNA in lung homogenate of mice adoptively transferred ( n = 9 CD40LG + T cells ) or not ( n = 8 CD40LG ) with in vivo primed CD4 + T cells in absence of conditioning and infected with the pathogen . Results are expressed in P . Murina / HPRT RNA copies . Mann - Whitney test . * * * P - value of the comparison = 0 . 0003 . Median ± IQR .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Quantitation of P. Murina rRNA in lung homogenate of mice adoptively transferred (n=9 CD40LG + T cells) or not (n=8 CD40LG) with in vivo primed CD4+ T cells in absence of conditioning and infected with the pathogen. Results are expressed in P.Murina/HPRT RNA copies. Mann-Whitney test. ***P-value of the comparison = 0.0003. Median ± IQR.


I Lung weight of experimental mice from ( H ) . Mann - Whitney test . * * P - value of the comparison = 0 . 0055 . Median ± IQR .

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I Lung weight of experimental mice from (H). Mann-Whitney test. **P-value of the comparison = 0.0055. Median ± IQR.


( A ) Livedo reticularis in the left abdominal lower quadrant after the first infusion in Pt 01 .

(A) Livedo reticularis in the left abdominal lower quadrant after the first infusion in Pt 01.


( B ) Small clump of MABs observed in the first preparation of MP before the infusion of Pt 01 .

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(B) Small clump of MABs observed in the first preparation of MP before the infusion of Pt 01.


( C ) Livedo reticularis in the left hand of Pt02 after the first infusion .

(C) Livedo reticularis in the left hand of Pt02 after the first infusion.


( D ) Brain MRI acquired one day after the MAB infusion showing acute small thalamic stroke in Pt03 . Axial diffusion - weighted imaging ( left ) and fluid - attenuated inversion recovery ( FLAIR ; right ) images show a focal spot of hyperintensity within the right thalamus consistent with acute stroke . ( E ) FLAIR MRI axial image obtained in Pt 03 one month after the acute stroke , showing the expected evolution of the right thalamic lesion .

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(D) Brain MRI acquired one day after the MAB infusion showing acute small thalamic stroke in Pt03. Axial diffusion-weighted imaging (left) and fluid-attenuated inversion recovery (FLAIR; right) images show a focal spot of hyperintensity within the right thalamus consistent with acute stroke.(E) FLAIR MRI axial image obtained in Pt 03 one month after the acute stroke, showing the expected evolution of the right thalamic lesion.


( A ) Haematoxilin and Eosin staining of muscle biopsies from Pt 01 , Pt 02 and Pt 03 performed 2 months after the last MAB infusion . Images show diffuse increase of connective tissue , atrophic and hypertrophic fibers , degenerating fibers , and diffuse centralization of nuclei .

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(A) Haematoxilin and Eosin staining of muscle biopsies from Pt 01, Pt 02 and Pt 03 performed 2 months after the last MAB infusion. Images show diffuse increase of connective tissue, atrophic and hypertrophic fibers, degenerating fibers, and diffuse centralization of nuclei.


( B ) Haematoxilin and Eosin staining of muscle biopsies from Pt 05 and Pt 06 performed one month before the first MAB infusion and 2 months after the last MAB infusion . Images show diffuse increase of connective tissue , atrophic and hypertrophic fibers , degenerating fibers , and diffuse centralization of nuclei . No evident differences were observed between biopsy performed after and before treatment .

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(B) Haematoxilin and Eosin staining of muscle biopsies from Pt 05 and Pt 06 performed one month before the first MAB infusion and 2 months after the last MAB infusion. Images show diffuse increase of connective tissue, atrophic and hypertrophic fibers, degenerating fibers, and diffuse centralization of nuclei. No evident differences were observed between biopsy performed after and before treatment.


( C ) Immunohistochemistry showing fetal myosin expression in muscle biopsy of DMD patients performed 2 months after the last MAB infusion . The staining was performed in tibialis anterior muscle for Pt 01 , Pt 02 and Pt 03 and in gastrocnemius muscle for Pt 05 and Pt 06 . A higher number of positive ( brownish staining ) fibers ( representing regenerating fibers ) were observed in Pt 02 , Pt 05 and Pt 06 .

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(C) Immunohistochemistry showing fetal myosin expression in muscle biopsy of DMD patients performed 2 months after the last MAB infusion. The staining was performed in tibialis anterior muscle for Pt 01, Pt 02 and Pt 03 and in gastrocnemius muscle for Pt 05 and Pt 06. A higher number of positive (brownish staining) fibers (representing regenerating fibers) were observed in Pt 02, Pt 05 and Pt 06.


( A ) Confocal immunofluorescence of muscle biopsy from Pt 01 ( tibialis anterior post - treatment ) stained with anti - dystrophin dys1 antibody ( which recognizes protein fragment encoded by exons 26 - 30 , green signal ) and anti laminin - 2 ( to delineate muscle fibers , red signal ) ; dapi identifies nuclei ( blue signal ) . In the left image only anti dystrophin staining is shown . No dystrophin positive fibers were observed . ( B ) Confocal immunofluorescence of muscle biopsy from Pt 02 ( tibialis anterior post - treatment ) stained with anti - dystrophin dys1 antibody ( green signal ) and anti lamininin - 2 ( red signal ) ; dapi identifies nuclei ( blue signal ) . In the left image only anti dystrophin staining is shown . Some fiber shows mild and discontinuous dystrophin staining .

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(A) Confocal immunofluorescence of muscle biopsy from Pt 01 (tibialis anterior post-treatment) stained with anti-dystrophin dys1 antibody (which recognizes protein fragment encoded by exons 26-30, green signal) and anti laminin-2 (to delineate muscle fibers, red signal); dapi identifies nuclei (blue signal). In the left image only anti dystrophin staining is shown. No dystrophin positive fibers were observed.(B) Confocal immunofluorescence of muscle biopsy from Pt 02 (tibialis anterior post-treatment) stained with anti-dystrophin dys1 antibody (green signal) and anti lamininin-2 (red signal); dapi identifies nuclei (blue signal). In the left image only anti dystrophin staining is shown. Some fiber shows mild and discontinuous dystrophin staining.


( C ) Immunofluorescence of muscle biopsy from Pt 05 taken before ( left , gastrocnemius ) and after treatment ( right , gastrocnemius ) stained with anti dystrophin dys2 antibody ( which recognizes exon 77 - 79 ) . The number and intensity of dystrophin - positive fibers is increased in the post - treatment biopsy . ( D ) Immunofluorescence of muscle biopsy from Pt 06 taken before ( left , gastrocnemius ) and after treatment ( right , gastrocnemius ) stained with anti dystrophin dys2 antibody . Few fibers show scattered dystrophin staining , without obvious differences between pre and post - treatment samples .

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(C) Immunofluorescence of muscle biopsy from Pt 05 taken before (left, gastrocnemius) and after treatment (right, gastrocnemius) stained with anti dystrophin dys2 antibody (which recognizes exon 77-79). The number and intensity of dystrophin-positive fibers is increased in the post-treatment biopsy.(D) Immunofluorescence of muscle biopsy from Pt 06 taken before (left, gastrocnemius) and after treatment (right, gastrocnemius) stained with anti dystrophin dys2 antibody. Few fibers show scattered dystrophin staining, without obvious differences between pre and post-treatment samples.


( E ) Results of western blot analysis involving dystrophin antibodies dys1 , Mandys18 and Manex46e ( recognizing respectively exon 26 - 30 , 17 - 35 , and 46 ) , of total protein extracts ( 20 µg ) obtained from post - treatment biopsy specimens of Pt 01 ( tibialis anterior muscle ) ; Ct was used as positive dystrophin control . Below , bands corresponding to myosin heavy chain are shown as loading control . No bands corresponding to full length dystrophin were observed . A schematic representation of the deleted portion of the dystrophin and the region recognized by used antibodies is depicted above the western blot . ( F ) Results of western blot analysis involving dystrophin antibodies Mandys18 and Manex46e , of total protein extracts ( 10 - 20 µg ) obtained from pre - treatment ( performed at time of diagnosis ) and post - treatment biopsy specimens of Pt 02 ( tibialis anterior muscle ) ; Ct was used as positive dystrophin control . Below , bands corresponding to myosin heavy chain are shown as loading control . One faint band corresponding to full length dystrophin is observed only in the post - treatment samples with both Mandys18 and Manex46e antibodies . A schematic representation of the deleted portion of the dystrophin and the region recognized by used antibodies is depicted above the western blot . ( G ) Results of western blot analysis involving dystrophin antibodies Mandys106 ( recognizing exon 43 ) and dys1 , of total protein extracts ( 80 µg , following protein concentration by Amicon Ultra - 0 . 5 centrifugal filter devices , Millipore ) obtained from pre - treatment and post - treatment biopsy specimens of Pt 05 ( gastrocnemius ) ; Ct was used as positive dystrophin control , CtDmd was used as dystrophin negative control . Below , bands corresponding to myosin heavy chain are shown as loading control . Bands corresponding approximately to full length dystrophin are observed in pre and post - treatment samples with both antibodies However , bands in post - treatment sample appeared higher in amount . A schematic representation of the dystrophin point mutation ( black vertical bar ) and the region recognized by used antibodies is depicted above the western blot . ( H ) Results of western blot analysis involving dystrophin antibodies Mandys106 and dys1 , of total protein extracts ( 80 µg , following protein concentration by Amicon Ultra - 0 . 5 centrifugal filter devices , Millipore ) obtained from pre - treatment and post - treatment biopsy specimens of Pt 06 ( gastrocnemius ) ; Ct was used as positive dystrophin control . Below , bands corresponding to myosin heavy chain are shown as loading control . Bands corresponding approximately to full length dystrophin are observed in pre and post - treatment samples with both antibodies Bands in pre - treatment sample appeared higher in amount as compared to post - treatment sample . A schematic representation of the dystrophin point mutation ( black vertical bar ) and the region recognized by used antibodies is depicted above the western blot .

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

(E) Results of western blot analysis involving dystrophin antibodies dys1, Mandys18 and Manex46e (recognizing respectively exon 26-30, 17-35, and 46), of total protein extracts (20 µg) obtained from post-treatment biopsy specimens of Pt 01 (tibialis anterior muscle); Ct was used as positive dystrophin control. Below, bands corresponding to myosin heavy chain are shown as loading control. No bands corresponding to full length dystrophin were observed. A schematic representation of the deleted portion of the dystrophin and the region recognized by used antibodies is depicted above the western blot.(F) Results of western blot analysis involving dystrophin antibodies Mandys18 and Manex46e, of total protein extracts (10-20 µg) obtained from pre-treatment (performed at time of diagnosis) and post-treatment biopsy specimens of Pt 02 (tibialis anterior muscle); Ct was used as positive dystrophin control. Below, bands corresponding to myosin heavy chain are shown as loading control. One faint band corresponding to full length dystrophin is observed only in the post-treatment samples with both Mandys18 and Manex46e antibodies. A schematic representation of the deleted portion of the dystrophin and the region recognized by used antibodies is depicted above the western blot.(G) Results of western blot analysis involving dystrophin antibodies Mandys106 (recognizing exon 43) and dys1, of total protein extracts (80 µg, following protein concentration by Amicon Ultra-0.5 centrifugal filter devices, Millipore) obtained from pre-treatment and post-treatment biopsy specimens of Pt 05 (gastrocnemius); Ct was used as positive dystrophin control, CtDmd was used as dystrophin negative control. Below, bands corresponding to myosin heavy chain are shown as loading control. Bands corresponding approximately to full length dystrophin are observed in pre and post-treatment samples with both antibodies However, bands in post-treatment sample appeared higher in amount. A schematic representation of the dystrophin point mutation (black vertical bar) and the region recognized by used antibodies is depicted above the western blot.(H) Results of western blot analysis involving dystrophin antibodies Mandys106 and dys1, of total protein extracts (80 µg, following protein concentration by Amicon Ultra-0.5 centrifugal filter devices, Millipore) obtained from pre-treatment and post-treatment biopsy specimens of Pt 06 (gastrocnemius); Ct was used as positive dystrophin control. Below, bands corresponding to myosin heavy chain are shown as loading control. Bands corresponding approximately to full length dystrophin are observed in pre and post-treatment samples with both antibodies Bands in pre-treatment sample appeared higher in amount as compared to post-treatment sample. A schematic representation of the dystrophin point mutation (black vertical bar) and the region recognized by used antibodies is depicted above the western blot.


( A ) Axial T1 - weighted images of the right thighs obtained in patients immediately before ( upper panel ) and 18 months after MAB infusion . Please note the progression of fatty degeneration and atrophy except for Pt 05 where no evident modifications can be detected . ( B ) Representative percentile of MRI quantitative parameters in transplanted patients compared to untreated patients . SIR ( left image ) and MVI ( right image ) of the quadriceps . Red line : Pt 01 ; dotted green line : Pt 02 ; dotted purple line : Pt 03 ; dotted dark blue line : Pt 05 ; dotted light blue line : Pt 06 ) . Empty dots correspond to pre - treatment measures . Orange dots correspond to post - transplantation measurement . The black line corresponds to the median , while gray dotted lines to 75 ° , 90 ° and 95 ° percentile . Please note the modification of the trend of Pt 05 after treatment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Axial T1-weighted images of the right thighs obtained in patients immediately before (upper panel) and 18 months after MAB infusion. Please note the progression of fatty degeneration and atrophy except for Pt 05 where no evident modifications can be detected. (B) Representative percentile of MRI quantitative parameters in transplanted patients compared to untreated patients. SIR (left image) and MVI (right image) of the quadriceps. Red line: Pt 01; dotted green line: Pt 02; dotted purple line: Pt 03; dotted dark blue line: Pt 05; dotted light blue line: Pt 06). Empty dots correspond to pre-treatment measures. Orange dots correspond to post-transplantation measurement. The black line corresponds to the median, while gray dotted lines to 75°, 90° and 95° percentile. Please note the modification of the trend of Pt 05 after treatment.


( C ) Representative quantile spline regressions of post - transplant trend amelioration of MRI quantitative parameters in Pt 05 . Upper left : SIR of the quadriceps . Lower left : SIR of thigh flexor muscles . Upper right : MVI of the quadriceps . Lower right : MVI of the semitendinosus muscles . Please note the significant modification of the patient trend ( red lines ) compared to median measurements obtained with 85 MRI examinations of untreated patients . Empty dots correspond to pre - treatment measures . Full dots correspond to post - transplantation measurement . The grey dotted line corresponds to the time of the first intra - arterial transplantation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 quantile spline regressions of post-transplant trend amelioration of MRI quantitative parameters in Pt 05. Upper left: SIR of the quadriceps. Lower left: SIR of thigh flexor muscles. Upper right: MVI of the quadriceps. Lower right: MVI of the semitendinosus muscles. Please note the significant modification of the patient trend (red lines) compared to median measurements obtained with 85 MRI examinations of untreated patients. Empty dots correspond to pre-treatment measures. Full dots correspond to post-transplantation measurement. The grey dotted line corresponds to the time of the first intra-arterial transplantation.


( A ) North Star scale measurement ( NSAA score ) plotted against age of ambulant DMD patients before , throughout and after MAB clinical trial . Arrows indicate time points of MAB infusion . Dotted line indicates the time period in which Pt05 spontaneously suspended steroids treatment without communication to parents and clinicians . Pt 02 and Pt 05 showed score stabilization throughout eight months of MAB infusions ; Pt05 showed stabilization even in the subsequent period , whereas Pt02 showed progressive deterioration until lost of ambulation . Pt 03 showed progressive deterioration throughout MAB infusion and lost ambulation soon after the end of the trial .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) North Star scale measurement (NSAA score) plotted against age of ambulant DMD patients before, throughout and after MAB clinical trial. Arrows indicate time points of MAB infusion. Dotted line indicates the time period in which Pt05 spontaneously suspended steroids treatment without communication to parents and clinicians. Pt 02 and Pt 05 showed score stabilization throughout eight months of MAB infusions; Pt05 showed stabilization even in the subsequent period, whereas Pt02 showed progressive deterioration until lost of ambulation. Pt 03 showed progressive deterioration throughout MAB infusion and lost ambulation soon after the end of the trial.


( B ) Six minute walking test ( meters ) plotted against age of ambulant DMD patients before , throughout and after MAB clinical trial . Arrows indicate time points of MAB infusion . Dotted line indicates the time period in which Pt05 spontaneously suspended steroids treatment without communication to parents and clinicians . Pt 02 and Pt 05 showed score stabilization throughout 8 months of MAB infusions ; Pt05 showed stabilization even in the subsequent period , whereas Pt 02 showed progressive deterioration until lost of ambulation . Pt 03 showed progressive deterioration throughout MAB infusion and lost ambulation soon after the end of the trial .

(B) Six minute walking test (meters) plotted against age of ambulant DMD patients before, throughout and after MAB clinical trial. Arrows indicate time points of MAB infusion. Dotted line indicates the time period in which Pt05 spontaneously suspended steroids treatment without communication to parents and clinicians. Pt 02 and Pt 05 showed score stabilization throughout 8 months of MAB infusions; Pt05 showed stabilization even in the subsequent period, whereas Pt 02 showed progressive deterioration until lost of ambulation. Pt 03 showed progressive deterioration throughout MAB infusion and lost ambulation soon after the end of the trial.


( C ) Time to run 10 meters ( time ) plotted against age of ambulant DMD patients before , throughout and after MAB clinical trial . Arrows indicate time points of MAB infusion . Dotted line indicates the time period in which Pt 05 spontaneously suspended steroids treatment without communication to parents and clinicians . Pt 02 and Pt 05 showed time stabilization throughout 8 months of MAB infusions ; Pt 05 showed stabilization even in the subsequent period , whereas Pt 02 showed progressive increase of time until lost of ambulation . Pt 03 showed progressive increase of time throughout MAB infusion and lost ambulation soon after the end of the trial .

(C) Time to run 10 meters (time) plotted against age of ambulant DMD patients before, throughout and after MAB clinical trial. Arrows indicate time points of MAB infusion. Dotted line indicates the time period in which Pt 05 spontaneously suspended steroids treatment without communication to parents and clinicians. Pt 02 and Pt 05 showed time stabilization throughout 8 months of MAB infusions; Pt 05 showed stabilization even in the subsequent period, whereas Pt 02 showed progressive increase of time until lost of ambulation. Pt 03 showed progressive increase of time throughout MAB infusion and lost ambulation soon after the end of the trial.


Panel A : viral and donor specific T - cell responses were measured by IFN - γELISpot at different time points : before the beginning of treatment ( PRE ) , prior to each infusion and 2 months after the fourth infusion . Infusions are indicated by arrows . Patient ' s peripheral blood mononuclear cells ( PBMC ) were challenged with irradiated autologous lymphoblastoid cell lines ( EBV line host ) , with cytomegalovirus glycin extract ( CMV antigen ) ( Gehrz et al , 1987 ) and with the following irradiated cells harvested from the donor : untreated mesoangioblasts ( MABdonor ) , MAB activated by 48 hour - exposure to 500 IU / mL IFN - γ ( γMABdonor ) , myotubes differentiated from MAB ( myotubesdonor ) and PBMC ( PBMCdonor ) . Polyclonal stimulation ( phytohemagglutinin , PHA ) was used as positive control . Donor PBMC were challenged with autologous targets as negative controls . Results are expressed as number of specific cells / 105PBMC and calculated according to the following formula : number of spots produced by patient ' s PBMC - number of spots produced by donor ' s PBMC . Results for patientsPt 01 , Pt 02 , Pt 03 , Pt 05 and Pt 06 are shown .

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Panel A: viral and donor specific T-cell responses were measured by IFN-γELISpot at different time points: before the beginning of treatment (PRE), prior to each infusion and 2 months after the fourth infusion. Infusions are indicated by arrows. Patient's peripheral blood mononuclear cells (PBMC) were challenged with irradiated autologous lymphoblastoid cell lines (EBV line host), with cytomegalovirus glycin extract (CMV antigen)(Gehrz et al, 1987) and with the following irradiated cells harvested from the donor: untreated mesoangioblasts (MABdonor), MAB activated by 48 hour-exposure to 500 IU/mL IFN-γ (γMABdonor), myotubes differentiated from MAB (myotubesdonor) and PBMC (PBMCdonor). Polyclonal stimulation (phytohemagglutinin, PHA) was used as positive control. Donor PBMC were challenged with autologous targets as negative controls. Results are expressed as number of specific cells/105PBMC and calculated according to the following formula: number of spots produced by patient's PBMC - number of spots produced by donor's PBMC. Results for patientsPt 01, Pt 02, Pt 03, Pt 05 and Pt 06 are shown.


Panel B : dystrophin specific T - cell responses were measured by IFN - γ ELISpot at two time points : before the beginning of treatment ( PRE , white bar ) and 2 months after the fourth infusion ( black bar ) . PBMC were challenged with a library of 15 - mer overlapping peptides covering the protein sequence ( pool 1 - 8 ) . Polyclonal stimulation ( phytohemagglutinin , PHA ) was used as positive control . The grey area represents the threshold calculated on a cohort of healthy donors . Results for Pt 01 , Pt 02 , Pt 03 , Pt 05 and Pt 06 are shown .

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

Panel B: dystrophin specific T-cell responses were measured by IFN-γ ELISpot at two time points: before the beginning of treatment (PRE, white bar) and 2 months after the fourth infusion (black bar). PBMC were challenged with a library of 15-mer overlapping peptides covering the protein sequence (pool 1-8). Polyclonal stimulation (phytohemagglutinin, PHA) was used as positive control. The grey area represents the threshold calculated on a cohort of healthy donors. Results for Pt 01, Pt 02, Pt 03, Pt 05 and Pt 06 are shown.


( A ) ELISA assa from brain lysates showing higher PTX3 expression in late embryonic and early postnatal brain , and lower in the adult brain . Data are presented as mean ± SEM . ( A ) ng PTX3 / mg of total proteins , E15 = 5 , 702 ± 0 . 365 , P0 = 4 . 904 ± 0 . 865 , P7 = 3 . 818 ± 0 . 530 , P14 = 2 . 140 ± 0 . 107 , P28 = 3 . 125 ± 0 . 525 , P90 = 2 . 212 ± 0 . 324 . One - way ANOVA analysis of variance followed by post hoc Tukey test : E15 vs P14 * * p = 0 . 001 , E15 vs P28 * p = 0 . 022 , E15 vs P90 * * p = 0 . 002 ; P0 vs P14 $ p = 0 . 013 , P0 vs P90 $ p = 0 . 016 ; 4 animals for each time

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ELISA assa from brain lysates showing higher PTX3 expression in late embryonic and early postnatal brain, and lower in the adult brain. Data are presented as mean±SEM. (A) ng PTX3/mg of total proteins, E15=5,702±0.365, P0=4.904±0.865, P7=3.818±0.530, P14=2.140±0.107, P28=3.125±0.525, P90=2.212±0.324. One-way ANOVA analysis of variance followed by post hoc Tukey test: E15 vs P14 **p=0.001, E15 vs P28 *p=0.022, E15 vs P90 **p=0.002; P0 vs P14 $p=0.013, P0 vs P90 $p=0.016 ; 4 animals for each time point


( B ) real time qPCR from brain lysates showing higher PTX3 expression in late embryonic and early postnatal brain , and lower in the adult brain . Data are presented as mean ± SEM ( B ) qPCR fold change normalized on E15 , E15 = 1 . 037 ± 0 . 154 , P0 = 0 . 711 ± 0 . 094 , P7 = 0 . 472 ± 0 . 0524 , P14 = 0 . 135 ± 0 . 007 , P28 = 0 . 168 ± 0 . 020 , P90 = 0 . 183 ± 0 . 029 . One - way ANOVA analysis of variance followed by post hoc Tukey test : E15 vs P7 * * * p = 0 . 0001 , E15 vs P14 * * * * p < 0 . 0001 , E15 vs P2 * * * * p < 0 . 0001 , E15 vs P90 * * * * p < 0 . 0001 ; P0 vs P14 $ $ $ p = 0 . 0007 , P0 vs P28 $ $ p = 0 . 001 , P0 vs P90 $ $ p = 0 . 002 ; 4 animals for each time

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(B) real time qPCR from brain lysates showing higher PTX3 expression in late embryonic and early postnatal brain, and lower in the adult brain. Data are presented as mean±SEM (B) qPCR fold change normalized on E15, E15=1.037±0.154, P0=0.711±0.094, P7=0.472±0.0524, P14=0.135±0.007, P28=0.168±0.020, P90=0.183±0.029. One-way ANOVA analysis of variance followed by post hoc Tukey test: E15 vs P7 ***p=0.0001, E15 vs P14 **** p<0.0001, E15 vs P2 ****p<0.0001, E15 vs P90 ****p<0.0001; P0 vs P14 $$$p=0.0007, P0 vs P28 $$p=0.001, P0 vs P90 $$p=0.002; 4 animals for each time point


Quantitation of PTX3 levels in pure astrocyte and neuronal cultures . ( C ) ELISA assay performed on astrocyte and neuronal culture mediu Only astrocytes release PTX3 ( ELISA , astro = 3 . 699 ± 1 . 129 , neu = N . D . , not detectable . 3 independent astrocyte cultures evaluated Data are presented as mean ±

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Quantitation of PTX3 levels in pure astrocyte and neuronal cultures. (C) ELISA assay performed on astrocyte and neuronal culture mediu Only astrocytes release PTX3 (ELISA, astro=3.699±1.129, neu=N.D., not detectable. 3 independent astrocyte cultures evaluated Data are presented as mean±SEM


Quantitation of PTX3 levels in pure astrocyte and neuronal cultures ( D ) rea time qPCR performed on astrocyte and neuronal lysates qPCR astro = 1 . 167 ± 0 . 288 ; neu = 0 . 148 ± 0 . 057 ; Mann - Whitney test * * p = 0 . 0095 . 2 independent neuronal cultures evaluated . Data are presented as mean ±

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Quantitation of PTX3 levels in pure astrocyte and neuronal cultures (D) rea time qPCR performed on astrocyte and neuronal lysates qPCR astro=1.167±0.288; neu=0.148±0.057; Mann-Whitney test **p=0.0095. 2 independent neuronal cultures evaluated. Data are presented as mean±SEM


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

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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 )

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

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

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

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

G) 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 B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR 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 B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O 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

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

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

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

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

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

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

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

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

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

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

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

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

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).


( A ) The intracellular distribution of DOR is modified by starvation . HeLa cells were transiently transfected with DOR and incubated for 1 h with DMEM ( basal ) , HBSS ( starvation ) , 50 μM chloroquine or 10 mM 3MA . The intracellular localization of DOR was analysed by immunofluorescence and is shown in red . The nuclei are shown in blue ( DAPI staining ) . Scale bars , 10 μm .

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

(A) The intracellular distribution of DOR is modified by starvation. HeLa cells were transiently transfected with DOR and incubated for 1 h with DMEM (basal), HBSS (starvation), 50 μM chloroquine or 10 mM 3MA. The intracellular localization of DOR was analysed by immunofluorescence and is shown in red. The nuclei are shown in blue (DAPI staining). Scale bars, 10 μm.


( B ) Endogenous DOR leaves the nucleus under starvation . C2C12 myoblasts were incubated for 30 min at 37 ° C with DMEM ( basal ) or HBSS ( starvation ) . DOR is shown in red and nuclei are in blue ( DAPI staining ) . Scale bars , 5 μm .

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(B) Endogenous DOR leaves the nucleus under starvation. C2C12 myoblasts were incubated for 30 min at 37°C with DMEM (basal) or HBSS (starvation). DOR is shown in red and nuclei are in blue (DAPI staining). Scale bars, 5 μm.


( C , D ) De ‐ activation of autophagy returns DOR to the nucleus . HeLa cells were transiently transfected with DOR and incubated for 1 h with DMEM ( basal ) or HBSS ( starvation ) . Cells were then incubated with normal DMEM for an additional hour ( starvation + GM ) . The intracellular distribution of DOR was determined by immunofluorescence . DOR is shown in red and the nuclei are in blue . Scale bars , 5 μm . Cell lysates were also obtained and western blot assays were performed with specific antibodies . 3MA , 3 ‐ methyladenine ; DAPI , 4 ′ , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; DMEM , Dulbecco ' s modified Eagle medium ; DOR , diabetes ‐ and obesity ‐ regulated gene ; HBSS , Hank ' s balanced salt solution ; LC3 , microtubule ‐ associated protein 1A / 1B ‐ light chain 3 .

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(C,D) De‐activation of autophagy returns DOR to the nucleus. HeLa cells were transiently transfected with DOR and incubated for 1 h with DMEM (basal) or HBSS (starvation). Cells were then incubated with normal DMEM for an additional hour (starvation+GM). The intracellular distribution of DOR was determined by immunofluorescence. DOR is shown in red and the nuclei are in blue. Scale bars, 5 μm. Cell lysates were also obtained and western blot assays were performed with specific antibodies. 3MA, 3‐methyladenine; DAPI, 4′,6‐diamidino‐2‐phenylindole; DMEM, Dulbecco's modified Eagle medium; DOR, diabetes‐ and obesity‐regulated gene; HBSS, Hank's balanced salt solution; LC3, microtubule‐associated protein 1A/1B‐light chain 3.


DOR localizes to autophagosomes when autophagy is activated . ( A ) Confocal images of HeLa cells transiently transfected with DOR and GFP ‐ LC3 , and incubated for 1h with DMEM , HBSS ( starvation ) , 2 μM rapamycin or HBSS containing 50 μM chloroquine . Nuclei were labelled with DAPI . Scale bars , 10 μm .

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DOR localizes to autophagosomes when autophagy is activated. (A) Confocal images of HeLa cells transiently transfected with DOR and GFP‐LC3, and incubated for 1h with DMEM, HBSS (starvation), 2 μM rapamycin or HBSS containing 50 μM chloroquine. Nuclei were labelled with DAPI. Scale bars, 10 μm.