For the in vivo mammary fat pad xenograft model illustrated in Fig. 2C and fig. S4E, 6-week-old female NOD/SCID mice were used. MDA-MB-231 LM2 cells (1 × 106) were harvested and resuspended in 50 μl of PBS and 50 μl of Matrigel. Cells were injected directly into the mammary fat pads of the mice, and tumor volumes were calculated by V = L × (W × ½)2, where L is length (longest dimension) and W is width (shortest dimension). For the in vivo lung metastasis xenograft model illustrated in Fig. 2G and figs. S4F and S6F, 6-week-old female BALB/c nude mice were used. MDA-MB-231 LM2 cells (1 × 105; stably expressing shTROJAN or shCtrl) were harvested in PBS and injected into the lateral tail vein in a total volume of 100 μl. For the in vivo bone metastasis xenograft model illustrated in Fig. 2H, 6-week-old female BALB/c nude mice were used. SCP2 cells (1 × 105; stably expressing shTROJAN or shCtrl) were harvested in PBS and injected into the left ventricle in a total volume of 100 μl. The mice were imaged for luciferase activity immediately after injection to exclude baseline bias. After the mice were euthanized, their lungs or livers were removed, imaged, and placed into 4% paraformaldehyde for paraffin embedding. For the bone metastasis xenograft models, the forelimb and hindlimb long bones of the mice were removed, fixed in 4% paraformaldehyde after 24 hours, decalcified (10% EDTA, 2 weeks), dehydrated through a graded alcohol series, embedded in paraffin, and stained with H&E.

The bioluminescence imaging shown in Fig. 2G was performed using the Lumazone imaging system (MAG BioSystems), and the bioluminescence imaging shown in Fig. 2H and figs. S4F and S6F was performed using the NightOWL LB 983 in vivo imaging system (Berthold Technologies). Relative bioluminescence signal quantitation was calculated by the respective imaging system software packages.

For biochemical analysis, the eyeballs of the mice were extracted, and blood samples were collected, placed at room temperature for 30 min, and centrifuged at 4500g for 15 min. The serum (supernatants) samples were collected for biochemical analysis using the automatic biochemical analyzer 8021A (URIT).

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