Fig. 6: EPN3-driven partial EMT relies on a TGFβ-based autocrine loop. | Nature Communications

Fig. 6: EPN3-driven partial EMT relies on a TGFβ-based autocrine loop.

From: A self-sustaining endocytic-based loop promotes breast cancer plasticity leading to aggressiveness and pro-metastatic behavior

Fig. 6

a RT-qPCR analysis of mRNA expression of TGFβ ligands (TGFβ1, TGFβ2) and receptors (TGFβR1, TGFβR2) in MCF10A-Ctr and -EPN3 cells. Mean relative mRNA expression compared with MCF10A-Ctr ± S.D. of nine independent experiments, each performed in technical triplicates. P value, Student’s t test two-tailed. b ELISA assay of secreted TGFβ1 in conditioned media from MCF10A-Ctr and -EPN3 cell cultures. Results are reported as pg ml−1 10−6 cells ± S.D. (n = 3, each performed on the medium of at least two independent dishes, each assayed in technical triplicates). P value, Student’s t test two-tailed. The same data are also reported in Supplementary Fig. 8A top for comparison between MCF10A-Ctr, -EPN3, -EPN1, and -TWIST. c Top, TCF4 or TGFβR1 were silenced in the indicated cells. Mock transfection was used as negative control. Cell lysates were IB with the indicated Ab. This panel was assembled from samples run on the same gel by splicing out the irrelevant lanes (as shown by the black lines). GAPDH, loading control. Right, MW markers. Relative protein levels of NCAD, ECAD and VIM of this blot were quantified together with other experiments (see Supplementary Fig. 8C for quantitation and statistics). Bottom, representative phase-contrast microscopy of MCF10A-EPN3 cells, mock, TCF4 KD or TGFβR1 KD. Bar, 250 µm. d RT-qPCR analysis of mRNA expression of the indicated genes in MCF10A-Ctr and -EPN3 cells that were mock-treated or subjected to TGFβR1 KD or TCF4 KD, and stimulated or not with TGFβ1 (5 ng ml−1, 48 h). Results are reported as relative mRNA expression compared with the MCF10A-Ctr mock-treated sample, mean ± S.D. (n ≥ 3, each performed in technical triplicates). P value, each pair Student’s t test, two-tailed. Black asterisks, statistical significance between KD samples vs. mock, not treated, and KD samples vs. mock, treated with TGFβ1, as indicated by the bar; red asterisks, statistical significance between matched MCF10A-EPN3 samples vs. -Ctr samples. e Proposed model of the EPN3-dependent activation of the EMT program in mammary epithelial cells. Source data are provided as a Source Data file.

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