1. Volume 24, Issue 11, Pages 1898-1912 (November 2016)
Therapeutic Potential of Immunoproteasome Inhibition in Duchenne Muscular Dystrophy 
Andrea Farini, Clementina Sitzia, Barbara Cassani, Letizia Cassinelli, Rosita Rigoni, Federica Colleoni, Nicola Fusco, Stefano Gatti, Pamela Bella, Chiara Villa, Filomena Napolitano, Rita Maiavacca, Silvano Bosari, Anna Villa, Yvan Torrente 
Molecular Therapy 
Volume 24, Issue 11, Pages (November 2016)
DOI: /mt
Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
i-proteasome evaluation of mdx and IFN-γ treated C57Bl mice. (a) RT-qPCR expression of PSMB5, PSMB8 and PSMB9 in muscles of mdx mice, indicated as fold increase versus C57Bl mice. (b) RT-qPCR expression of IFN-γ in in muscles of mdx and C57Bl mice. (c, c') WB expression of PSMB5, PSMB8 and PSMB9 of freshly isolated and IFN-γ-treated myoblasts isolated from C57Bl muscles. (d) RT-qPCR expression of PSMB5, PSMB8, and PSMB9 of freshly isolated and IFN-γ-treated myoblasts obtained from C57Bl muscles. IFN-γ, interferon gamma; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; PSMB9, 20S proteasome – β1i; PSMB8, 20S proteasome – β5i.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Immunological characterization of mdx mice. (a) Weight and cellularity of spleens from 6–8 weeks old mdx, scid-mdx and C57Bl mice. (b) Representative FACS plots showing gating strategy used for analysis of splenic myeloid cell population. Numbers in quadrants indicate the percentage of each cell type. Graphs show cumulative absolute counts of myeloid cells subsets in the spleen of mdx, scid/mdx and C57Bl mice. (c) Graphs report cell numbers of splenic CD4 and CD8 T cells in mdx, scid/mdx, and C57Bl mice. Representative FACS plots show (d) immunophenotype of CD4+ and CD8+ T cells and (e) the amount of IFN-γ producing CD4+ and CD8+ cells recovered from spleen of mdx and C57Bl mice. Data are representative of 2–3 independent experiments with n = 3–18 animals/group. IFN-γ, interferon gamma; FACS, fluorescent-activated cell sorting.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 2
Immunological characterization of mdx mice. (a) Weight and cellularity of spleens from 6–8 weeks old mdx, scid-mdx and C57Bl mice. (b) Representative FACS plots showing gating strategy used for analysis of splenic myeloid cell population. Numbers in quadrants indicate the percentage of each cell type. Graphs show cumulative absolute counts of myeloid cells subsets in the spleen of mdx, scid/mdx and C57Bl mice. (c) Graphs report cell numbers of splenic CD4 and CD8 T cells in mdx, scid/mdx, and C57Bl mice. Representative FACS plots show (d) immunophenotype of CD4+ and CD8+ T cells and (e) the amount of IFN-γ producing CD4+ and CD8+ cells recovered from spleen of mdx and C57Bl mice. Data are representative of 2–3 independent experiments with n = 3–18 animals/group. IFN-γ, interferon gamma; FACS, fluorescent-activated cell sorting.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 3
Characterization of lymphocyte infiltrates in the muscles of mdx mice. (a) Representative FACS plots of CD4 and CD8 T cells. Numbers in quadrants indicate the percentage of each cell type. Plots were gated on CD45 positive population. Graphs showed absolute numbers of T cells infiltrating the muscles of mdx, scid-mdx and C57Bl mice. Counts were normalized for the weight (expressed in grams, gr) of digested tissue. (b) Representative FACS plots showed immunophenotype of CD4+ and CD8+ T cells recovered from muscle tissue of mdx and C57Bl mice. Graphs showed cumulative frequency of naive (CD62L+CD44−), central memory (CD62L+ CD44+) and effector (CD62L-CD44+) T subsets. Data are representative of 2–3 independent experiments with n = 3–8 animals/group. IFN-γ, interferon gamma; FACS, fluorescent-activated cell sorting.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 4
ELISPOT analysis of mdx and ONX-0914 treated mdx mice. (a) Representative images of mdx splenocytes treated with dystrophin peptides against exon 18 of dystrophin gene, mdx splenocytes alone and mdx splenocytes supplemented with Concanavalin A (used as positive control). (b) Histogram shows the number of spots in two regions corresponding to exons 2–12 and 18–23 of dystrophin gene in mdx and ONX-0914 treated mdx mice. Data are representative of three independent experiments with n = 3–13 animals/group. Animals of 6 weeks of age were treated 5 weeks/2 times per week with ONX-0914, and then sacrificed. ELISPOT, enzyme-linked immunosorbent spot.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 5
Analysis of ONX-0914 treatment on splenic immune cells. (a) Characterization of weight and cellularity of spleens of ONX-0914 treated and untreated mdx, scid/mdx and C57Bl mice. Quantification of cellular subpopulations for the expression of CD11b and F4/80 (b), of inflammatory CD4+ and CD8+ lymphocytes (c) and T cell subsets (naive, central memory, and effector memory) (d) in spleens of treated and untreated mdx, scid/mdx and C57Bl mice. (e) Quantification of circulating Foxp3+CD25+ CD4+ Treg in treated and untreated C57Bl and mdx mice. (f) Representative FACS plots show the amount of IFN-γ producing CD4+ and CD8+ cells recovered from spleen of treated and untreated mdx mice. Data are representative of 2–3 independent experiments with n = 3–18 animals/group. Six-weeks-old mice were treated 5 weeks/2 times per week with ONX-0914, and then sacrificed. FACS, fluorescence-activated cell sorting.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

8. Figure 6
Amelioration of dystrophic phenotype in ONX-0914 treated mdx mice. (a) H&E and (b) AM images of TA and QA of ONX-0914-treated and untreated mdx mice. Histogram represents the percentage of fibrotic area in muscle section, calculated as collagen positive area in AM (c), and percentage of necrotic fiber (d). (e) Dystrophin staining of QA of ONX-0914-treated and untreated mdx mice highlight dystrophin re-expression. (f) WB analysis of dystrophin expression in mdx mouse (used as negative control), while TA and QA treated with ONX-0914 showed the expression of truncated isoform (120 KDa) instead of the full-length dystrophin (427 KDa). (g) CPK values of C57Bl and mdx mice throughout 5 weeks of ONX-0914 treatment. (h) Histogram of tetanic force of TA muscles of ONX-0914-treated and untreated C57Bl, mdx and scid/mdx mice. Immunostaining (i) and WB analysis (j) of β-sarcoglycan in QA of ONX-0914-treated and untreated mdx mice, showed partial rescue of DGC complex. Data are representative of 2–3 independent experiments with n = 3–13 animals/group. Six-weeks-old mice were treated 5 weeks/2 times per week with ONX-0914, and then sacrificed. CPK, creatine phospho-kinases; DGC, dystrophin glycoprotein complex; H&E, Haematoxilin and Eosin; TA, Tibialis Anterior; QA, Quadriceps; WB, western blot.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

9. Figure 7
Effect of ONX-0914 treatment on muscle immune infiltrates of mdx mice. The immunohistochemistry (IHC) showed reduction of CD45+ cells and MHC-I+ myofibers in ONX-0914 treated mdx mice (a). The quantification of MHC-I+ myofibers confirmed a statistically significant decrease of these myofibers in treated mdx mice (a″). FACS analysis of muscles of ONX-0914 treated and untreated C57BL and mdx mice reveals a decrease of CD45+ cell number (a′); significant reduction of CD4+ and CD8+ T lymphocytes (b); significant increase of CD4+ and CD8+ naive T cells and decrease of CD8+ effector cells (c) in ONX-0914 treated mdx mice. In these experiments scid/mdx were used as negative control. (d) Foxp3 IHC staining of ONX-0914 treated and untreated mdx muscles. (e) Foxp3+ cell number was significantly higher after treatment, as indicated in the histogram. (f) Quantitative PCR experiments performed on treated and untreated mdx muscles. Relative expression values revealed significant differences in the expression of TNF-α, IL-1β and IFN-γ (g) WB analysis revealed higher expression of IL-1β in QA and TA treated muscles related to untreated ones. Mouse Cell lysate—whole cell (hepatocytes) was used as positive control. Data are representative of 2–3 independent experiments with n = 3–13 animals/group. Six-week-old mice were treated 5 weeks/2 times per week with ONX-0914, and then sacrificed. IFNγ, interferon gamma; IL-1β, interleukin-1 β; TA, Tibialis Anterior; TNF-α, tumor necrosis factor alpha; QA, Quadriceps; WB, western blot. FACS, fluorescence-activated cell sorting.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions