Journal: bioRxiv

Article Title: Tuning of granulopoietic signaling by de novo designed agonists

doi: 10.1101/2023.11.25.568662

Figure Lengend Snippet: Representative western blot images of intracellular levels of phospho-STAT3 (Tyr705) and STAT3 (A) and phospho-STAT5 (Tyr694) and STAT5 (B) proteins after treatment of NFS-60 cells with 1 nM of different designs (saturating conditions) for 5 min (left pane) or 30 min (right pane). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) staining was used as a loading control. Three biological replicates of this experiment showed the same trend . (C, D) The pSTAT3 and pSTAT5 levels were quantified after 5 min and 30 min from those replicates and are represented in (C) and (D), respectively. Shown is the fold change to the GAPDH normalized rhG-CSF signal. (E, F) To probe the effect of the agonist designs (100 ng/ml) and rhG-CSF (10 ng/ml) on healthy donoŕs CD34 + HSPCs, proliferation assays were performed without (E) or with the addition of 50 ng/mL SCF and 20 ng/mL IL-3 (F). Ori0 without SCF and IL-3 was tested at three concentrations 100 ng/mL (diamond), 10 ng/mL (triangle-right), and 1 ng/mL (triangle-left). Shown is the mean (points) and standard deviation (shades) of three parallel replicates. (G, H, I) Additionally, we performed CFU assays of healthy donoŕs CD34 + HSPCs incubated on semi-solid medium supplemented with corresponding cytokines and design agonists. Shown is the fold change to rhG-CSF of the quantified colony-forming units (CFU) of granulocytes (G-CFU) (G), granulocyte-macrophages (GM-CFU) (H), and macrophages (M-CFU) (I). The circles represent the obtained values for each condition of three independent experiments with two parallel replicates each. The indicated p-values were estimated by an ordinary one-way ANOVA followed by a Tukey HSD test.

Article Snippet: Human CD34 + cells were isolated from the bone marrow mononuclear cell fraction of two healthy donors by Ficoll density gradient centrifugation with subsequent magnetic bead separation using the Human CD34 Progenitor Cell Isolation Kit (Miltenyi Biotech Germany; #130-046-703).

Techniques: Western Blot, Staining, Control, Standard Deviation, Incubation

Journal: Orphanet Journal of Rare Diseases

Article Title: Impaired osteoblast and osteoclast function characterize the osteoporosis of Snyder - Robinson syndrome

doi: 10.1186/s13023-015-0235-8

Figure Lengend Snippet: Characterization of the bone and osteoblast pathology. A . Photograph of the bone biopsy. B . Steady state SMS mRNA levels relative to GAPDH expression in cultured fibroblasts and osteoblasts. The patient’s cells did not differ significantly from controls. Data were derived by qRT-PCR analysis of 3 independent extractions of total RNA. C . Immunoblot showing steady state SMS protein expression in patient and control osteoblasts. ß-tubulin is shown as a loading control. D . Graph showing steady state SMS protein levels in the patient and control hBMSCs relative to ß-tubulin levels; there was no significant difference. The data are based on 3 independent experiments for each cell line. E - J . Immunofluorescent detection of SMS protein subcellular distribution in unaffected (E-G) and Patient II-1 (H-J) hBMSCs. SMS protein is shown in red and the nucleus is shown in blue. K . Graph quantifying immunoblot detected steady state SMS protein levels in the cytoplasm and nuclei of patient and control hBMSCs. The cytoplasmic expression was normalized to β-tubulin expression and the nuclear expression to p84 expression. L . Polyamine quantification in fibroblasts and osteoblasts. Note that the patient hBMSCs have a more striking imbalance of spermidine and spermine levels than do the patient fibroblasts, * p < 0.05, *** p < 0.005. M . Osteogenic potential of bone marrow stromal cells (hBMSCs) isolated from Patient II-1 sample is markedly lower than that of an unaffected control (cnt). The hBMSCs were seeded in triplicates (6x10 4 /12-well) and either kept untreated (-) or treated (+) with osteogenic differentiation media (see ) for 18 days. After the treatment, cells were fixed and were stained with Alizarin Red S to check for calcium deposition, a marker of osteogenic differentiation.

Article Snippet: We also measured spermine and spermidine levels in cultured human bone marrow stromal cells (hBMSCs) and fibroblasts using the Biochrom 30 polyamine protocol and assessed the osteogenic potential of hBMSCs.

Techniques: Expressing, Cell Culture, Derivative Assay, Quantitative RT-PCR, Western Blot, Control, Isolation, Staining, Marker