Fig 2: Malignant megakaryocytes express great levels of CXCR1 and CXCR2. (A) Representative sections from the bone marrow of wild-type and Gata1low littermates immunostained with antibodies per CXCR1 and CXCR2, showing the level of these receptors in endothelial cells, osteoblasts, megakaryocytes, and neutrophils (indicated with arrows). Semiquantitative estimates (-, +, or ++) of the intensity of the staining in the different cells are provided on the top left. Original magnification 40×. Representative sections stained only with the secondary antibodies, are shown in Figure S6 (Supplementary Materials), as negative controls. (B) Percentage of megakaryocytes expressing high levels of CXCR1 and CXCR2. The proinflammatory cytokines were quantified as described in Figure S2B (Supplementary Materials) on five randomly selected areas per femur from three–four mice per experimental group. Statistical analysis was performed by t-test, and statistically significant p values among groups are indicated within the panels. Abbreviations: CXCR1 and 2: CXC motif chemokine receptor 1 and 2; WT: wild-type; MKs: megakaryocytes.

Fig 3: In the lung from Gata1low mice, treatment with the P-SEL inhibitor reduces the content of both TGF-ß1 and CXCL1 and increases that of GATA1 of the megakaryocytes while those with either the TGF-ß1 or the CXCR12 inhibitor reduce the levels of CXCL1 only.A) Immunohistochemical staining for TGF-ß1 and CXCL1 of consecutive lung sections from representative Gata1low mice treated either with vehicle (one mouse representative of the vehicle group for all the treatments) or with the P-SEL (day 12 and 40), TGF-ß1 (5 and 10 weeks) or CXCR1/2 (20 or 37 days) inhibitor, as indicated. Magnification 400x. B,C) Quantification of the TGF-ß1 (B) and CXCL1 (C) content of lungs from Gata1low mice treated either with vehicle or inhibitors for P-SEL, TGF-ß1 or CXCR1/2, as indicated. Data are presented as Mean (±SD) and as values per individual mice (each symbol a different mouse) and were analyzed by one-way Anova. P values statistically significant are indicated within the panels. D) Double immune-fluorescence staining with anti-GATA1 (green) and CD42b (red as a marker of MK) of lung sections from representative Gata1low mice treated either with vehicle or inhibitors for either P-SEL (day 12 and 40), TGF-ß1 (5 and 10 weeks) or CXCLR1/2 (20 or 37 days), as indicated. Nuclei were highlighted with DAPI (blue). Magnification 400x. The signal captured in the individual channels are presented in Figure S10. E) Total number of MK (CD42bpos cells) and of MK expressing detectable levels of GATA1 in the lungs from Gata1low mice treated either with vehicle or the P-SEL, TGF-ß1 or CXCR1/2 inhibitor, as indicated. Data are presented as Mean (±SD) and as values per individual mice (each symbol a different mouse) and were analyzed by one-way Anova. P values statistically significant are indicated within the panels.

Fig 4: Development of fibrosis in the lung from Gata1low mice is prevented by genetic ablation of P-selectin and is rescued by treatments with inhibitors of either P-selectin, TGF-ß or CXCR1/2.A) H&E, Reticulin and Masson’s trichrome staining of consecutive sections of lungs from representative WT, Gata1low, P-selnull and P-selnullGata1low mice (all 12-15 months old), as indicated. Magnification 40x. Results observed in multiple mice per experimental group are presented and analyzed in detail in Figure 2 and Table 1. B, E and H) H&E, Reticulin and Masson’s trichrome staining of consecutive sections from lungs of representative Gata1low mice treated with either vehicle or the P-SEL antibody for 5, 12 or 40 days (B), with either vehicle or the TGF-ß inhibitor for 5 or 10 weeks (E) or either with vehicle or the CXCR1/2inhibitor for 20 or 37 days. H), as indicated. Magnification 200x. C, F and I) Number of Gata1low mice (in percent of the total) expressing Grade 0, 1, 2 or 3 of inflammation when treated with either vehicle or the P-SEL inhibitor C), or when treated either with vehicle or the TGF-ß (F) or either with vehicle or the CXCR1/2 inhibitor (I). Results of the vehicle groups at different time points were pooled, because similar. D, G, J) Level of fibrosis detected by reticulin or Masson staining in consecutive lung sections from mice treated either with vehicle or with the P-SEL inhibitor (D), either with vehicle or the TGF-ß inhibitor (G). or either with vehicle or the CXCR1/2 inhibitor (J). Data are presented as Mean (±SD) and as data per individual mouse (each dot a single mouse). Statistical analyses were performed were by one-way ANOVA and p values statistically significant between groups are indicated within the panels.