Journal: Stem Cells (Dayton, Ohio)
Article Title: Calcium and Calcineurin-NFAT Signaling Regulate Granulocyte-Monocyte Progenitor Cell Cycle via Flt3-L
Figure Lengend Snippet: Calcineurin-nuclear factor of activated T cells (NFAT) inhibition results in increased granulocyte–monocyte progenitors (GMP) proliferation in vitro, due to changes in the expression of cell cycle control genes in FLT-3L-differentiated progenitors. (A): Heat maps reflecting changes in the expression of genes related to cell cycle regulation, transcriptional regulation of differentiation, and hematopoiesis are shown. cKIT + -enriched cells were cultured in hematopoietic stem cell (HSC) medium stimulated with of rmFlt3-L (300 ng/ml) in the presence or absence of Cyclosporine A (CsA) (24 and 48 hours). Data represent three replicates per time point and treatment. Cells from 10 mice were pooled for each biological replicate. (B–D): Differential expression of Cdk4, Cdk6 , and Cdkn1a ( p21 ) mRNA within progenitor populations. Sorted HSCs (lin − , cKit + , Sca-1 + , CD34 + , Flt3 − ), multipotent progenitors (MPPs) (lin − , cKit + , Sca-1 + , CD34 + , Flt3 + ), common myeloid progenitors (CMPs) (lin − , cKit + , Sca-1 − , CD34 + , CD16/32 − ), and GMPs (lin − , cKit + , Sca-1 − , CD34 + , CD16/32 + ) were cultured for 24 hours in HSC medium in the presence of Flt3-L (300 ng/ml) before the expression of main cell cycle regulatory genes was measured by qRT-PCR. Data represent at least five independent experiments, bone marrow (BM) from 15 mice pooled for each. One-way analysis of variance (ANOVA) with Turkey multiple comparisons test was used, *, p
Article Snippet: Reverse transcription was carried out using high-capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems), or with SuperScript III First Strand Synthesis System for RT-polymerase chain reaction (PCR) (Invitrogen).
Techniques: Inhibition, In Vitro, Expressing, Cell Culture, Mouse Assay, Quantitative RT-PCR