Treatment of bone marrow cells with Sonic Hedgehog induces stress BFU-E formation. (A) Bone marrow cells were preincubated overnight in Iscove modified Dulbecco medium plus 5% fetal calf serum supplemented with (+Shh) or without (Control) Sonic Hedgehog. Afterward, the cells were plated in methylcellulose media containing either Epo (3 U/mL) alone or Epo + BMP4 (15 ng/mL), and BFU-E were scored. (B) Real-time RT-PCR analysis of the expression of BMP4 by bone marrow cells cultured in the presence or absence of Shh. (C) Bone marrow cells were preincubated for 24 hours with Shh as described in panel A and then plated in methylcellulose media containing Epo or Epo + Noggin (200 μg/mL), and BFU-E were scored. (D) RT-PCR analysis of BMP4 expression in the spleen of WBB6F1 Kit^W/Wv recipient mice transplanted with CD45.1 donor bone marrow cells. The vertical white line between time 0 and 7 days has been inserted to indicate a repositioned gel lane. (E) Analysis of BMP4 expression by CD45.1⁺ donor cells in the spleen of Kit^W/Wv mice after bone marrow transplantation. (Left) BMP4 is shown in red and CD45.1 in green in a low power (original magnification ×20) analysis of BMP4 expression. Overlap between the 2 signals is shown in yellow. SA indicates splenic artery; RP, red pulp; and WP, white pulp. (Right) Spleen sections from additional mice at higher power (original magnification ×40). BMP4 is shown in red, CD45.1 in green, and the overlap in signals as yellow. All assays were done in triplicate and are representative of 2 independent experiments. Significant differences are indicated in the figure. Slides were viewed with an Olympus BX61Epi Fluorescence microscope (Olympus, Center Valley, PA) using a UPlanF1 lens at 40×/0.75 NA and Slow Fade Gold antifade agent (Invitrogen, Carlsbad, CA). Images were acquired using an Olympus DP71 camera (Olympus) and were processed with DP-BSW Basic software for the DP71 camera and Adobe Photoshop imaging software (Adobe, San Jose, CA).