Description of CUX1 knockdown mouse models and characterization of MDS in Cux1^midmice. (A) Cux1 has cell type–specific expression levels. Cells were sorted as follows from the BM of wild-type C57BL/6 mice: LT-HSC (LSK⁺/CD34⁻/Flt3⁻), ST-HSC (LSK⁺/CD34⁺/Flt3⁻), MPP (LSK⁺/CD34⁺/Flt3⁺), CMP (Lin⁻/Sca1⁻/c-Kit⁺/CD34⁺/CD16/32^low), MEP (Lin⁻/c-Kit⁺/Sca1⁻/CD34⁻/CD16/32⁻), and GMP (Lin⁻/c-Kit⁺/Sca1⁻/CD34⁺/CD16/32^high). The following cells were sorted from the spleen: granulocytes (CD11b⁺/Gr1⁺), monocytes (CD11b⁺/Gr1⁻), B cells (B220⁺), and T cells (CD3⁺). qRT-PCR for Cux1 mRNA was performed. The data represent the mean ± SEM from 4 biological replicates. *Student t test, P < .05. (B) The Cux1 genomic locus is shown with all 7 RefSeq mRNA isoforms. shRNA (Cux1^low and Cux1^mid) targeting the indicated exons (red arrows) were used to generate transgenic mice. (C) A representative immunoblot for CUX1 protein in thymocytes isolated from Cux1^low, Cux1^mid, and Ren mice after 7 days of dox. The mean ± SD level of residual CUX1 protein quantified from 4 biological replicates for Cux1^low was 12% ± 9% and 54% ± 17% for Cux1^mid. (D) Cux1 mRNA level in sorted populations from Cux1^mid and Cux1^low BM compared with Ren littermate control. One representative replicate of 3 biological replicates is shown. LSK are Lin⁻/c-Kit⁺/Sca1⁺), and myeloid progenitors are Lin⁻/Sca1⁻/c-Kit⁺. (E) At 6 to 10 weeks of age, Cux1^mid and Ren littermate control mice were started on continuous dox treatment and monitored for up to 18 months. Complete blood counts of mice at 8 and 18 months of dox are shown. (F) Flow cytometric analysis of peripheral blood (PB) samples for monocyte (CD11b⁺/Gr1⁻) and granulocyte (CD11b⁺/Gr1⁺) populations at 8 and 18 months of dox treatment. (G) Spleen weights after 18 months of dox. The mean ± SD is shown. *P ≤ .05, **P ≤ .01, Wilcoxon rank test. (H-P) Representative morphology images are shown for mice after 18 months of dox. (H) Wright-Giemsa staining of the peripheral blood in (Hi) Ren and (Hii-iii) Cux1^mid mice. (Hii) Cux1^mid mice have an increase in circulating mature granulocytes (arrowheads). (Hiii) A granulocyte with pseudo Pelger-Huet anomaly is shown (yellow arrowhead). Red blood cells in Cux1^mid mice have increased Howell-Jolly bodies (arrow) and increased reticulocytes with basophilic stippling (black arrowheads). (Ii-ii) H&E staining of the bone marrow of aged Cux1^mid mice shows a marked increase in megakaryocytes. (Iiii) At high power, clusters of megakaryocytes (arrowheads) with dysplastic features can be appreciated, including micromegakaryocytes, nuclear hypolobation, and condensed, hyperchromatic nuclei. (Ji-ii) H&E staining shows an expansion of the red pulp at the expense of the white pulp. The white pulp is outlined by a yellow dashed line for clarity. (Jiii) High-power analysis of Cux1^mid spleens illustrates increased megakaryocytes (arrowheads) with some micromegakaryocytes with condensed, hyperchromatic nuclei. (K-L) Additional examples of peripheral blood granulocytes with pseudo Pelger-Huet anomaly. (M) Neutrophil with hypersegmentation. (N) A representative giant platelet is shown (arrow). (O) Touch preparation of Cux1^mid spleen reveals erythroid dysplasia as evidenced by binucleated erythroblasts (arrowheads) and abnormal nuclear contours (arrow). (P) H&E staining shows a myelomonocytic cell infiltrate (dashed yellow line) in the liver of Cux1^mid mice. Images were taken with a Zeiss Axioskop microscope. Chr, chromosome; H&E, hematoxylin and eosin; mRNA, messenger RNA; PB, peripheral blood; SD, standard deviation; SEM, standard error of the mean.