Microenvironmental bioavailability of pro-inflammatory cytokines rather than their serum levels correlates with develop-ment of myelofibrosis in Gata1low mice

Figure S1. Gating used to identify endothelial cells (ECs), mesenchymal cells (MSCs), osteoblasts (OBCs), megakaryocytes (MKs) and neutrophils (Neu) in the bone marrow from a wild type mouse. In order to correctly estimate the frequency of all the cell populations, the monocellular cell suspension used for the analyses were prepared by carefully crushing the femurs of the mice (see [33] and [34] for detail). Figure S2. (A) Example of the computer assisted image analysis used to quantify the level of fibrosis in bone marrow sections stained by Reticulin (black fibers) from representative wild-type (WT) and Gata1low mice. The ImageJ program-assisted quantification process includes selection of the color channel (black histogram, Hue), determination of the threshold (Saturation histogram) and quantification of the areas which exceed the threshold (in %) (Brightness histogram). (B) Example of the computer assisted image analysis used to quantify the number of megakaryocytes positive per CXCR1 in in bone mar-row sections from wild-type (WT) and Gata1low mice. In this case, the conditions to quantify the signal in wild-type and Gata1low mice were set separately. The ImageJ program-assisted quantification process includes selection of the cell type, determination of the threshold per cell (Saturation histogram) and quantification of the number of cells which exceed the threshold (Brightness histogram). Magnification 40x. Figure S3. A) Median optical slice of a confocal stack microscopy obtained with a 63x objective. The blue arrow indicates a GFP signal present inside nucleus (shown as tridimensional image in movie 1), whereas the red arrow indicates a cell were the GFP signal is localized in the cytoplasm (shown as tridimensional image in movie 2). DAPI and GFP signals are shown separately and combined. B) Hematoxylin-eosin staining (left) and fluorescent microscopy (right) of two repre-sentative sections from the bone marrow of one huCD34-GFPH2B mouse. The GFP staining labels either the nuclear areas of cells with small size (1, 2 and 3) or, very rarely, the cytoplasm of large cells (4), possibly macrophages. Slides were counter stained with DAPI. Magnification 40x. Figure S4. Hematoxylin/Eosin (H&E) and reticulin staining and TGF-β1, LCN2 and CXCL1 immunostaining of bone marrow sections from representative wild-type (WT) and Gata1low littermates showing the fibrosis and increased cytokine bioavailability in microenvironment of the mutant mice. Results are representative of those observed in 12-month old mice randomly selected from male and female, at least were analyzed 6 mice each staining. The arrows indicate individual megakaryocytes in wild-type mice and megakaryocyte clusters in Gata1low mice. Mag-nification 40x. Similar results were previously published in Dumbar et al, 2021[11]. Figure S5. The bone marrow from Gata1low mice contain great levels of CXCR1. High levels of CXCR1 and CXCR2 in bone marrow sections from Gata1low mice. A) Representative sections from the bone marrow of wild-type and Gata1low littermates immunostained with antibodies per CXCR1 and CXCR2. Magnification 40x. B) Computer assisted quantification of the content of the CXCR1 and CXCR2 in 5 randomly selected areas per femur from three-four mice per experi-mental group. Statistical analysis was done by t-test and statistically significant p values among groups are indicated within the panels. Similar results were reported in Dunbar et al[11]