Figure Supplementary 1: Characterization of ER+ breast cancer tumour mass dormancy model suitable for shRNA screen.

(a) Kaplan-Meier analysis of bone metastasis-free survival in a DBM cells xenograft (n = 12 mice). (b) Representative bioluminescence images showing bone metastasis progression in DBM xenograft (top), and quantification of BLI signal in bone metastatic lesion (n = 5 limbs with metastatic lesions) (bottom). (c) Kaplan–Meier analysis of bone metastasis-free survival in a BoM2 cell xenograft (n = 17 mice). (d) Representative bioluminescence images showing bone metastasis progression in BoM2 xenograft (top), and quantification of bone metastatic lesion BLI signal (bottom) (n = 10 limbs with metastatic lesions). (e) CGH analysis of DBM and parental cells. Genetic gains and losses in DBM cells, for each chromosome, are depicted with red and green color bars respectively. (f) Quantification of ex vivo BLI signal in bones isolated during homing (n = 8 limbs), latency (n = 7 limbs), metastasis (n = 8 limbs). Two-tailed Mann Whitney test. (g) Quantification of osteolythic area from X-ray scans in bones isolated during homing (n = 3 limbs) and metastasis (n = 5 limbs). Two-tailed Mann–Whitney test. Experiment was performed once. (h) Analysis of bone samples with detected micrometastatic lesions or single DTC by means of human ER_ IHC during homing (i) Quantification of phospho-P38-positive cells in latent lesions (n = 20 ROIs from 4 limbs) and metastatic lesions (n = 25 ROIs from 3 limbs). Two-tailed Mann-Whitney test (left). Representative image of phospho P38 staining in lesions during latency or metastatic phase (right). Scale bar, 50 µm. (j) Quantification of phospho-SMAD2-positive cells in latent lesions (n = 21 ROIs from 4 limbs) and metastatic lesions (n = 42 ROIs from 3 limbs). Two-tailed Mann-Whitney test. Experiment was performed once. Panels (b) and (d) show data as mean ± SEM, and panels (f), (g) and (i-j), as whisker plots: mid-line, median; box, 25 – 75 percentile; whisker, min-max. ns (nonsignificant) P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Statistics source data and P values are provided in Supplementary Table 9.