Supplementary Figure 4: Controls for Vimentin specific detection, pRb-detection in DTCs, Dendra2 photoconversion and NR2F1 expression in PyM tumors.

(A) Gray scale image of mock tail vein injected (no T-HEp3 cells, only PBS) Foxn1 nu/nu mouse lung stained for human vimentin and DAPI. Vimentin antibody showed no reaction with mouse lung tissue. Boxed area in 40x indicates higher magnification shown in 100x. Scale bar 25 μm (40x) and 10 μm (100x). (B) Representative images and quantification of T-HEp3 DTCs in mouse lungs at day 10 after injection and 5 days cis-platin treatment (see Fig 8A), stained for p-Rb (green), human vimentin (red) and DAPI (blue). Graph shows mean + SD, n = 3 mice, >500 cells per group; one tailed Student’s t test. (C) Frozen section of PyMT-Dendra2 PT photo converted with UV channel for 15sec (see dashed line). Scale bar 250 μm. (D) Representative images of a spontaneous MMTV-PyMT-Dendra2 (green) lung micro-metastasis stained for macrophage marker CD45 (red) and DAPI (blue). No significant green Dendra2 signal was detected in CD45 positive macrophages. Asterisks indicate cytoplasmic aggregates of Dendra2 protein observed in MMTV-PyMT-Dendra2 cells, as in Fig 8G-H. Scale bar 10 μm; n = 3 mice. (E) Increase in red pixel intensity of 34 label retaining (LRC) and 34 not label retaining (NLRC) PyMT-Dendra2 lung DTCs measured using MetaMorph. Graph LRC: fold increase normalized to red pixel intensity before photo conversion of slide. Graph NLRC: absolute red pixel intensity in arbitrary units, no red pixel intensity before PC. Cells marked with # are shown in Figure 8H. (F) Quantification of cells positive for NR2F1 in PyMT-Dednra2 primary tumor (PT) and spontaneous lung DTCs (see Fig 8E-I). Bars show mean + SD. n = 3 mice, 10 HPF per PT, 187 lung DTCs.