Journal: Science signaling

Article Title: Inflammation induces stress erythropoiesis through heme-dependent activation of SPI-C

doi: 10.1126/scisignal.aap7336

Figure Lengend Snippet: (A) Schematic of the LPS-zymosan model of sterile inflammation. (B) Splenocytes were harvested at the indicated time points and plated at a concentration of 1 × 105 cells per well in the presence of GDF15, BMP4, SCF, SHH, and EPO at low O2 to induce the formation of stress BFU-Es. Stress BFU-Es were scored after 5 days by staining with benzidine. Data represent the mean ± SEM, one-way ANOVA with Dunnett’s multiple comparison test, n = 4 to 11 mice per time point. (C) Flow cytometry analysis of splenocytes was performed at 0 and 36 hours after treatment with zymosan. Cells were gated on Kit+ cells, and frequencies of CD34+ and CD133+ populations are shown in representative images. n = 3 to 5 mice per time point. (D and E) RNA was isolated from splenocytes at the indicated time points after zymosan injection, and relative expression of Gdf15 and Bmp4 compared to 18S was determined by quantitative PCR. Data represent the mean ± SEM, one-way ANOVA with Dunnett’s multiple comparison test, n = 3 to 10 mice per time point. (F) Splenocytes harvested from mice at the indicated time points after zymosan treatment were plated under conditions to induce stress BFU-Es and stained with benzidine and counted after 5 days. Data represent the mean ± SEM, one-way ANOVA with Dunnett’s multiple comparison test, n = 3 to 9 mice per time point. (G) Protein was isolated from kidneys at the indicated time points after zymosan treatment and Western blotted for HIF-2α and β-actin. HIF-2α abundance relative to β-actin was calculated using ImageJ. Data represent the mean ± SEM, one-way ANOVA with Dunnett’s multiple comparison test, n = 3 mice per time point. (H) Quantification of Epo expression relative to 18S in RNA isolated from kidneys and abundance of EPO in serum at the indicated time points after zymosan treatment. Data represent the mean ± SEM, one-way ANOVA with Dunnett’s multiple comparison test, n = 2 to 5 mice per time point (mRNA) and 2 to 8 mice per time point (protein). *P < 0.05, **P < 0.01, and ***P < 0.005.

Article Snippet: Briefly, SEEM is composed of Iscove’s modified Dulbecco’s media containing 10% fetal bovine serum, insulin (10 μg/ml), holo-transferrin (200 μg/ml), 2 mM L-glutamine, ciprofloxacin (10 μg/ml), 1% bovine serum albumin (BSA), 2-mercaptoethanol (7 μl/liter), GDF15 (30 ng/ml, Biomatik), BMP4 (15 ng/ml, R&D Systems), sonic hedgehog (SHH) (25 ng/ml, GoldBio), and stem cell factor (SCF) (50 ng/ml, Gold-Bio).

Techniques: Concentration Assay, Staining, Flow Cytometry, Isolation, Injection, Expressing, Real-time Polymerase Chain Reaction, Western Blot