Antagomir-10b can be directly delivered to tumor cells in vitro and can inhibit cell motility and invasiveness.
(a) Real-time RT-PCR of miR-10b in cultured 4T1 cells treated with PBS or antagomir-10b.
(b) Immunoblotting of Hoxd10 in 4T1 cells treated with PBS or antagomir-10b. Full-length blots and molecular weight markers are presented in Supplementary Figure 6.
(c) Transwell migration assay and Matrigel invasion assay of 4T1 cells treated with PBS or antagomir-10b.
(d) Growth curves of 4T1 cells treated with PBS or antagomir-10b.
(e) Real-time RT-PCR of Hoxd10 in cultured 4T1 cells transfected with Hoxd10 siRNA or control oligonucleotides.
(f) Transwell migration assay and Matrigel invasion assay of control siRNA- or Hoxd10 siRNA-transfected 4T1 cells that are treated with either antagomir-10b or the vehicle (PBS). A representative experiment is shown in triplicate along with s.e.m. in a, and c–f.

Antagomir-10b treatment does not affect late stages of the metastatic process.
(a) Schematic representation of the antagomir administration schedule for mice with tail vein injection of 4T1 cells.
(b) Real-time RT-PCR of miR-10b in livers of mice with tail vein injection of 4T1 cells and subsequent treatment with PBS or antagomir-10b. Data are presented as mean ± s.e.m. (n = 6 mice in each group; each data point represents the mean expression value of triplicates of the sample from one mouse).
(c,d) Bright field imaging and H&E staining of the lungs (c) and number of visible lung metastases (d) in PBS- or antagomir-10b-treated mice at day 19 after tail vein injection of 4T1 cells. Scale bars, 800 µm for bright field imaging; 200 µm for H&E staining. Data in d are presented as mean ± s.e.m. (n = 6 mice in each group).

‘Sponge’-mediated silencing of miR-10b in tumor cells is sufficient to inhibit metastasis.
(a) Real-time RT-PCR of miR-10b in 4T1 cells infected with the miR-10b sponge or control sponge. A representative experiment is shown in triplicate along with s.e.m.
(b) Weight of primary mouse mammary tumors formed by 4T1 cells infected with the miR-10b sponge or control sponge.
(c,d) Bright field imaging and H&E staining of the lungs (c) and number of visible lung metastases (d) in mice bearing 4T1 cells infected with the miR-10b sponge or control sponge, at 4 weeks after orthotopic implantation. Arrows indicate lung metastases. Scale bars, 800 µm for bright field imaging; 200 µm for H&E staining. Data in b and d are presented as mean ± s.e.m. (n = 5 mice in each group).

Toxicity assessment following intravenous delivery of antagomir-10b in normal mice.
(a) Real-time RT-PCR of miR-10b in normal Balb/c mice after treatment with PBS or six doses of 50 mg/kg antagomir-10b or antagomir-10b_mm. Data are presented as mean ± s.e.m. (n = 6 mice in each group; each data point represents the mean expression value of triplicates of the sample from one mouse).
(b) Total body weight was measured twice a week during the study.
(c) H&E-stained sections of liver samples. Arrows indicate Kupffer cell macrophages. The circle indicates occasional lobular lymphocytes. Scale bars, 30 µm.
(d,e) White blood cell (d) and lymphocyte (e) count. Data in b, d, and e are presented as mean ± s.e.m. (n = 5 mice in each group).

The metastasis-suppressing effect of antagomir-10b is sequence-specific.
(a) Real-time RT-PCR of miR-10b in primary breast tumors (left panel) and livers (right panel) of 4T1 tumor-bearing mice treated with antagomir-10b or antagomir-10b_mm. Data are presented as mean ± s.e.m. (n = 6 mice in each group; each data point represents the mean expression value of triplicates of the sample from one mouse).
(b) Immunoblotting of Hoxd10 in primary breast tumors of 4T1 tumor-bearing mice treated with antagomir-10b or antagomir-10b_mm. SE: short exposure; LE: long exposure. Full-length blots and molecular weight markers are presented in Supplementary Figure 6.
(c) Primary tumor weight of 4T1 tumor-bearing mice treated with antagomir-10b or antagomir-10b_mm, at 4 weeks after orthotopic implantation.
(d,e) Bright field imaging (d, scale bars, 800 µm) and H&E staining (e, scale bars, 200 µm) of the lungs from 4T1 tumor-bearing mice treated with antagomir-10b or antagomir-10b_mm, at 4 weeks after orthotopic implantation. Arrows indicate lung metastases.
(f,g) Number of visible lung metastases (f) and metastasis index (= metastasis number divided by primary tumor weight, g) in 4T1 tumor-bearing mice treated with antagomir-10b or antagomir-10b_mm, at 4 weeks after orthotopic implantation. Data in c, f, and g are presented as mean ± s.e.m. (n = 9 mice per group).