Mx1-Cre–mediated inactivation of Rb and E2f8 in HSCs leads to hemolysis. (A) Prussian blue staining of tissue sections from mice with the indicated genotypes. Yellow arrows indicate a positive macrophage in the spleen, a positive Kupffer cell in the liver, and a positive proximal tubular cell in the kidney, whereas the green arrow indicates a hepatocyte with weak Prussian blue staining. Insets represent enlarged images of representative positive cells. Scale bar indicates 20 μm. (B) Representative images of new methylene blue staining of blood smears prepared from mice with the indicated genotypes. Black arrows show typical reticulocytes on blood smears. The inset shows an enlarged image of a representative reticulocytes. Scale bar indicates 10 μm. (C) Percentages of reticulocytes quantified from panel B. A minimum of 200 RBCs were counted for analysis (n ≥ 3 mice/genotypic group). (D) RBC life spans were assessed by biotin labeling and cytometric analysis (n ≥ 3 mice/genotypic group). (E) RBC deformability was assessed by ektacytometry (n ≥ 3 mice/genotypic group). (F) Representative confocal images of peripheral blood stained with rhodamine-phalloidin. Yellow arrows show spherocytes and green arrows show echinocytes. (G) Quantifications of data from panel F for abnormal erythrocytes (spherocytes and echinocytes). Reticulocytes and leukocytes were excluded from the quantification by counterstaining the peripheral blood with SYTOX Green. Images were acquired on a Zeiss LSM 510 confocal microscope and analyzed with LSM Image Browser Version 4.2 (Carl Zeiss). Scale bar indicates 5 μm. A minimum of 200 cells were counted for analysis (n = 3 mice/genotypic group). (H) Representative flow cytometric profiles using CD71 Abs and Thiazole Orange and percentages of reticulocytes (CD71⁺TO⁺) and CD71⁺ erythrocytes (CD71⁺TO⁻) in the peripheral blood. Numbers are mean percentages ± SD (n ≥ 3 mice/genotypic group).