Journal: Science translational medicine

Article Title: Muscarinic acetylcholine receptor regulates self-renewal of early erythroid progenitors

doi: 10.1126/scitranslmed.aaw3781

Figure Lengend Snippet: (A) Chemical structure of muscarinic acetylcholine receptor antagonist oxyphenonium bromide (OB). (B) Highly purified murine BFU-Es were isolated as lin−Ter119−CD16/CD32−Sca-1−CD41−c-Kit+CD71/Cd24a10%low population from murine fetal liver (14, 15, 19) and cultured with DMSO or 100 mM OB, and cell numbers were counted from days 0 to 9. The means and SD of three measurements from distinct samples are shown. P value was calculated using two-way ANOVA (original data are in data file S3). (C) Chemical structure of CHRM4 selective antagonist PD102807. (D) IC50 values from protein binding assay for PD102807 on indicated receptors. (E) Purified murine BFUEs were cultured with DMSO or 3 μM PD102807, and cell numbers were counted from days 0 to 9. The means and SD of three measurements from distinct samples are shown. P value was calculated using two-way ANOVA analysis (original data are in data file S3). (F) Purified murine BFU-Es were cultured with DMSO or 100 μM OB, and cells were stained with anti-Ter119 antibody at the end of culture and analyzed with flow cytometry. The means and SD of the percentage of Ter119+ cells in three measurements from distinct samples are shown. P value was calculated using the one-tailed t test. (G) Purified murine BFU-Es were cultured with DMSO or 100 μM OB, and cells were plated on methylcellulose medium. BFU-E colonies were counted on day 9 of colony formation assay, and the means and SD of BFU-E colonies in nine measurements from distinct samples are shown. P value was calculated using the one-tailed t test. (H) Purified murine BFU-Es were cultured with DMSO or 100 μM OB, and RNA-seq was performed on cultured cells. The x axis represents the ratio of each gene’s expression in BFU-Es cultured with OB relative to BFU-Es cultured with DMSO. The y axis represents the cumulative fraction and is plotted as a function of the relative expression (x axis). “BFU-E genes” represent a group of 533 genes most markedly down-regulated during erythroid differentiation from the BFU-E to the CFU-E stage (data file S2) (14). “All genes” represent all the genes expressed in BFU-E, as reported previously (15). P value was calculated using the Kolmogorov-Smirnov test. (I) The means and SD of relative expression of Kit in three measurements from distinct samples extracted from RNA-seq data displayed in (H). P value was calculated using the one-tailed t test. (J) The means and SD of relative expression of Zfp36l2 in three measurements from distinct samples extracted from RNA-seq data displayed in (H). P value was calculated using the one-tailed t test.

Article Snippet: Reagents BFU-E culture media, StemSpan serum-free expansion medium (SFEM) (09650) and StemSpan SFEM II (09655), and methylcellulose medium, MethoCult SF M3436 (03436), were purchased from STEMCELL Technologies.

Techniques: Purification, Isolation, Cell Culture, Protein Binding, Staining, Flow Cytometry, One-tailed Test, Colony Assay, RNA Sequencing, Expressing

Journal: Scientific Reports

Article Title: Autophagy confers DNA damage repair pathways to protect the hematopoietic system from nuclear radiation injury

doi: 10.1038/srep12362

Figure Lengend Snippet: ( A,B ) Mice in the treatment group were given 4 mg/kg of rapamycin by i.p. injection five times every other day before total body irradiation, whereas mice in the control group were injected with carrier DMSO. After irradiation, peripheral blood counts were performed at the indicated time points. In the rapamycin-treated group, white blood cell, lymphocyte, red blood cell, and platelet numbers were markedly higher than those of the control group soon after irradiation ( A ), but only white blood cell and lymphocyte counts were higher than those of the control for the long period post-irradiation ( B ). ( C ) Expression of γH2A.X was measured by flow cytometry in total bone marrow cells, lineage-positive cells, lineage-negative cells, and LSK cells of the two mouse groups following 24 h ex vivo incubation after whole body irradiation of the mice. DNA damage was significantly reduced in the rapamycin-treated group (right panel). Left panels are representative flow histograms of the treatment groups. ( D ) Rapamycin improved bone marrow progenitors’ colony formation under radiation exposure. Mice were pretreated with carrier or rapamycin for five times before γ-irradiation. Hematopoietic mononuclear cells were then collected from the bone marrow of the sacrifice mice at day 1 after 0 Gy, 1 Gy and 3 Gy by Ficoll gradient centrifugation. Clonogenic progenitors were determined in methylcellulose medium using 6 × 10 3 bone marrow mononuclear cells per 35 mm dishes and incubated for 7 days. The number of colonies containing more than 50 cells was determined. Colony numbers in the rapamycin-treated group were significantly increased after the radiation exposure. ( E ) LSK cells (Lin − Sca-1 + c-Kit + ) were sorted with fluorescent-activated cell sorting followed by magnetic-activated cell sorting for lineage-negative cells. Representative LSK sorting gates are shown (left and middle panels). The percentage number of LSK cells, expressed as a percentage against the number of total bone marrow mononuclear cells, was higher in the rapamycin-treated group than in the control group at day 3 after 5 Gy whole body irradiation (right panel). Data are mean ± SD from at least three independent experiments. n ≥ 5, *p < 0.05 and **p < 0.01.

Article Snippet: Clonogenic progenitors were determined in methylcellulose medium (Stem Cell Technologies, Alameda, USA) using 6 × 10 3 bone marrow mononuclear cells per dishes (35 mm) and incubated in a humidified atmosphere with 5% CO 2 at 37 °C for 7 days.

Techniques: Injection, Irradiation, Control, Expressing, Flow Cytometry, Ex Vivo, Incubation, Gradient Centrifugation, FACS