Extended Data Figure 4 : Cell density and regulation of PGR and HER2 signalling.

From: Early dissemination seeds metastasis in breast cancer

Extended Data Figure 4

a, PGR expression silenced in tumours can be re-activated in culture and re-silenced in vivo. b, PGR re-expression only occurs in TUBO cells grown at low density and high density after frequent medium change. c, Downregulation of PGR in early lesion cells cultured in a transwell assay next to primary tumour cells suggests the existence of a secreted factor passing through the membrane of the transwell insert and downregulating Pgr mRNA (left) and protein (right). d, T47D cells exposed to conditioned medium from TUBO cells display reduced PGR mRNA (left) and protein (right). e, Exosomes derived from the cell culture medium of TUBO cells grown at high density (exosome fraction) induce downregulation of PGR in T47D cells. f, miRNA sequencing to identify PGR regulating miRNAs. Left, top 10 upregulated miRNAs in Her2-overexpressing cells (MM3MG–Her2) compared to control (MM3MG). Middle, top 10 expressed miRNAs in TUBO cells and TUBO-cell-derived exosomes. Right, miRNAs predicted by the miRANDA web software to regulate Pgr g, Among all candidate miRNAs only miR-30a-5p and miR-9-5p induce downregulation of Pgr mRNA in T47D cells. h, Downregulation of PGR in T47D cells treated with miR-30a-5p and miR-9-5p. i, Expression of miR-30-5p in early lesions and primary tumour samples compared to mammary glands from 8-week-old BALB/c mice. j, Density induced upregulation of HER2 in TUBO cells grown at low or high density and early lesions compared to primary tumour samples (left) and progesterone-responsiveness of low-density TUBO cells (right). Note that levels of HER2, PGR, the glucocorticoid (GR), mineralocorticoid (MR) and androgen receptors (AR) are regulated by progesterone in a dose-dependent manner except ER-α (see related result in Extended Data Fig 1d). km, TUBO cells grown at low density and exposed to progesterone or PIPS migrated more (k, l) and produced more spheres (m), similar to cells derived from early lesions (see Fig. 2). **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001(Student’s t-test); data are mean ± s.d. For gel source data, see Supplementary Fig. 1.