Fig. 6

Iron is responsible for S Phase entry and cyclin E1 induction during B-cell proliferation. a Microarray analysis of global gene expression in control and iron-deficient B cells that were unstimulated or stimulated with anti-IgM (10 μg/ml) or LPS (2 μg/ml) for 48 h. Pathway analysis was conducted to map genes that were associated with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. b B cells cultured and stimulated as in a were cocultured with 10 μM BrdU for the last 45 min, stained with anti-BrdU and 7AAD, and analyzed by flow cytometry. The outlined areas represent the percentage of cells in S phase (with BrdU incorporation during DNA replication). c The mRNA levels of cyclin D2, cyclin E1, and Bcl-xL were measured by qRT-PCR in control and iron-deficient B cells that were cultured and stimulated as described in a. d Immunoblot analysis of cyclin E1, Bcl-xL, and phos-RelA in splenic B cells cultured in normal medium or in the presence of DFO (20 μM) or DFO with FAC (50 μM) under the indicated conditions for 48 h. e Splenic B cells from wild-type C57BL/6 mice in low-DFO conditions (10 µM) were infected with cyclin E1 overexpression lentivirus, and BrdU incorporation was analyzed by flow cytometry following anti-IgM (10 μg/ml) or LPS (2 μg/ml) stimulation for 48 h. f Statistics for the percentage of B cells proceeding through DNA replication and entering S phase. g Cyclin E1 and cyclin D2 mRNA were checked by qRT-PCR in control and cyclin E1-overexpression B cells. h qRT-PCR of cyclin E1 mRNA expression in splenic B cells isolated from control and iron-deficient mice immunized with DNP-KLH or TNP-LPS. The data are representative of two independent experiments. All graphs with error bars indicate the mean ± SEM, *P < 0.05, **P < 0.01, Student’s t-test