Fig 2: Pentraxin (PTX)3 expression in skeletal muscle biopsies of mdx mice at different ages. Representative Western blot (WB) of PTX3 in TAs of 10 weeks (10w), 3 months (3m), 5m, 7m C57Bl mice and in TAs of 10w, 9m, 14m, and 18m mdx mice. Data from densitometric analysis are expressed as PTX3/vinculin ratio in arbitrary units in the lower panels. One-way ANOVA with Tukey’s multiple comparisons test: *p < 0.05; **p < 0.01; ***p < 0.001 (A). In the lateral panels, the scheme representing PTX3 expression, in C57Bl and mdx mice, according to ages (B). Representative WB of PTX3 in TA of 10w, 5m, and 9m C57Bl mice and age-matched mdx mice. Data from densitometric analysis are expressed as the ratio of PTX3/vinculin in arbitrary units in the lateral panels. Student’s t-test: *p < 0.05; **p < 0.01 (C). Each experiment was performed in triplicate wells. All values are expressed as the mean ± SD.

Fig 3: Proteomic and inflammatory features of dsyferlinopathic skeletal muscle mice are modulated following ONX-0914 treatment.Proteomic analysis of IP subunits (A); alarmins and inflammatory mediators (PTX3 and GPx1) (B); MAPK kinases and AKT1/2/3 and their phosphorylated isoforms (C) in psoas of 6 m BlAJ, 12 m BlAJ and BlAJ+ONX, 12 m C57Bl mice. Psoas stained with isolectin (in green), PTX3 (in magenta) with nuclei stained in DAPI (blu) (scale bar: 10 µm) and its magnification (scale bar: 10 µm) with white arrows indicating cells co-expressing PTX3 and isolectin. The histogram represents the counting of PTX3 fluorescence (D). Immunohistological staining of skeletal muscles for Iba1 (in red) and CD206 (in green). Nuclei were counterstained with DAPI (blue). Scale bar: 50 µm. Quantification of macrophage percentages. Mac1 were stained with Iba1+ and Mac2 with CD206+ in 6 m BIAJ, 12 m BlAJ and BlAJ+ONX, 12 m C57Bl skeletal muscles (E). Data are presented as mean ± SD of n = 3 independent experiments with n = 3–6 animals/group. One-way ANOVA, Tukey multiple comparison test for WB and non-parametric test followed by Kruskal–Wallis test for PTX3+ cells’ and macrophages’ counting: *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Fig 4: Modulation of proteins in cardiac tissues of mdx mice at different ages following ONX-0914 treatment. Representative images of pentraxin (PTX)3 and PMSB8 immunohistochemistry staining of cardiac tissues showing PTX3- and PSMB8-positive endothelial cells (arrowheads) in cardiac tissues from 9 months (9m) and 18m mdx mice. Image magnifications: 10 × (scale bar: 200 mm) and 40 × (scale bar: 50 mm) (A). Representative Western blot (WB) in cardiac tissues of untreated and ONX-0914-treated 9m mdx mice for Toll-like receptor (TLR)2, TLR4, PTX3 (B); receptor for advanced glycation end-products (RAGE), high-mobility group box (HMGB)1, S100ß, and interleukin (IL)-33; collagen I, collagen VI, matrix metalloproteinase (MMP)9, and TRAF-6 (C); tumor necrosis factor (TNF)-a, poly(ADP-ribose) polymerase (PPAR)g, MYD88, RelB, nuclear factor (NF)-?B, ATG-7, and LC3B (D). Data from densitometric analysis are expressed as the ratio of different proteins versus vinculin (B) and actin (C,D) in arbitrary units in the lateral panels. Student’s t-test: **p < 0.01; ***p < 0.001 (B); *p < 0.05 (C); *p < 0.05; ***p < 0.001 (D). Each experiment was performed in triplicate wells. All values are expressed as the mean ± SD.

Fig 5: PTX3 reverses the PARPi effect on the VM formation. (A) Western blot analysis of PTX3 in the SUM159 cells after 24 h of treatment with BAY-11-7082 (2.5 µM). (B) Cells were treated for 72 h with olaparib (20 µM) and a human recombinant PTX3 was added for 24 h (1 µg/mL). Representative pictures were taken at 24 h and the tube formations were quantified. Experiments were realized at least three times and the data were expressed as mean ± SEM. *** p < 0.001.