(A) Effects of MELK knockdown on anchorage-independent growth of BBC cells in soft agar. The left and middle panels show crystal violet staining, and bright-field images of the colonies respectively. The bar graphs indicate the means ± SD for three experiments. (B) Effects of MELK knockdown on the growth of BBC cells in vivo. MDA-MB-468 and MDA-MB-231 cells carrying tet-shMELK were orthotopically implanted into the mammary fat pads of nude mice. The recipient mice were divided into two groups: one group of mice was given doxycycline-supplemented drinking water on the second day of injection for the duration of the experiment, while the other group of mice was maintained without doxycycline. The histogram indicates tumor volume measured 7 weeks after treatment. Data are means ± SEM (n ≥ 6). (C and D) Effects of MELK knockdown on established tumors arising from implantation of basal (C) or luminal (D) breast cancer cells. Mice bearing orthotopic tumors arising from the indicated cells carrying tet-shMELK were divided into two groups, with one group of mice receiving doxycycline, and the other maintained without doxycycline. Tumor volumes were measured on the indicated days after the administration of doxycyline. Data are means ± SEM (n ≥ 8). (E and F) Effects of MELK inhibition on tumor growth. Mice with tumors developed from basal (E) or luminal (F) breast cancer cells, were treated once daily with vehicle (0.5% methycellulose) or OTSSP167 (5 mg/kg). Tumor volumes were measured on the indicated days. Data are means ± SEM (n ≥ 8). (G) Knocking out Melk in mice. Indicated tissues were harvested from wild type or Melk−/− adult mice, and homogenized in RIPA lysis buffer. Lysates were subjected to immunoblotting. Total lysate of human breast cancer cell line MDA-MB-231 was used as a control. (H) Loss of Melk has no obvious impact on the development of immune system. Cells were isolated from bone marrow (BM), spleen and thymus, and subjected to flow cytometric analysis. Note that CD11b+/Gr1+ is a marker for neutrophils, CD11b+/Gr1− for monocytes, B220 for B cells, CD3, CD4 and CD8 for T cells. (I) Bone marrow was collected from wild type (wt) and Melk−/− (KO) mice and stained for the indicated cell populations. LSK: Lin−Sca1+ ckit+; LT-HSC(long-term hematopoietic stem cells): LSK CD150+CD48−; ST-HSC(short-term hematopoietic stem cells): LSK CD150−CD48−; CMP(Common myeloid progenitor): Lin−cKit+Sca1−IL7Ra−CD34+FcRg−; GMP(Granulocyte-macrophage progenitors): Lin−cKit+Sca1−IL7Ra−CD34+FcRg+; MEP(Megakaryocyte-erythrocyte progenitors): Lin−cKit+Sca1−IL7Ra−CD34−FcRg−; CLP(Common lymphoid progenitors): Lin−cKitmidSca1midIL7Ra+. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, Student's t test.