TPO increases HIF-1α expression. (A) After 24 hours of growth factor deprivation, UT-7/TPO cells were stimulated with 100 ng/mL hTPO for various time periods, nuclear extracts were prepared, and HIF-1α levels were analyzed by Western blotting. For an internal control, the membranes were reprobed with an anti-TFIIH antibody. (B) Bone marrow cells were prepared from BDF-1 mice and sca-1⁺/c-kit⁺/Gr-1^- cells were selected by cell sorting. The cells were cultured with or without 100 ng/mL hTPO for 24 hours; the cells were then fixed and stained with an anti-HIF-1α primary antibody and an Alexa488-conjugated secondary antibody (green). Cells were counterstained with DAPI (4,6 diamidino-2-phenylindole) to visualize nuclear DNA (blue). An example of the predominant staining pattern is shown. Images were obtained with a Leica DMLS microscope system (Wetzlar, Germany). Immunofluorescent images were obtained using SPOT RT software (Diagnostic Instruments, Sterling Heights, MI). The objective lens used was a Leica N plan, 40×/0.65; the camera used was Diagnostic Instruments, model 2.2.1 (Diagnostic Instruments).