Down-regulation of Kit expression by Epo.

<p>A: Kit and EpoR expression were studied in the 633, 663 and 812 cells grown continuously in the presence of either Epo (1 U/mL) or SCF (100 ng/mL) or a combination of Epo (1 U/mL)+SCF (100 ng/mL). Whole cell lysates were subjected to Western blot analysis with antibodies directed against Kit, EpoR and β-actin as a loading control. Western blots are from a representative experiment. The membrane was exposed in an Imager, and the resulting signal was quantified using the ImageGauge software package (Fuji, Paris, France). Values were normalized to β actin expression. The fold change in Kit expression between SCF-cultured cells and either Epo or Epo+SCF-cultured cells is indicated under Kit immunoblotting. B: Representative diagram of flow cytometry analysis showing Kit membrane expression in cells cultured with SCF (100 ng/mL; black line) or Epo+SCF (dotted line). Control IgG profile is shown in grey. The table indicates the mean fluorescence intensity (MFI)±SD of four independent experiments. C: AG490 inhibits the down-regulation of Kit by Epo. Cells were cultured for 48 hrs in a medium containing 10% FBS, 1 U/mL Epo and in the presence or absence (-) of AG490 (10 µM). Representative Western blot analysis of whole cell lysates with antibodies directed against Kit, Stat5 and phosphorylated Stat5. β-actin was used as loading control. The fold change in Kit expression between AG490-treated and untreated cells is indicated under Kit immunoblotting. Representative diagram of flow cytometry analysis showing Kit membrane expression in 663 and 812 cells cultured with Epo and treated (black line) or not (dotted line) with AG490 (10 µM) for 48 hrs. Control IgG profile is shown in grey. The table indicates the mean fluorescence intensity (MFI)±SD of positive cells in three independent experiments. D: Cells were cultured for 24, 48 and 72 hrs in the presence of a combination of Epo (1 U/mL)+SCF (100 ng/mL) or Epo (0.1 U/mL)+SCF (100 ng/mL) and viable cells were numbered. Data are mean±SD of five experiments, each performed in duplicate. E: The regulation of Kit expression is transcriptional: RT-PCR analysis of <i>Kit</i> and <i>EpoR</i> transcripts in 633, 663 and 812 cells cultured in the presence of either Epo (1 U/mL) or SCF (100 ng/mL). cDNAs were amplified with specific primers for <i>Kit</i>, <i>EpoR</i> or <i>gapdh</i> as control.