Fig. 4

Malignant MDCs transfer H19 LncRNA through secreting exosomes. A Sftpc-expressing AT2 cells sorted from lung metastatic tumors (tumor) or their peritumoral tissues (peri) of 16-week-old MPG-ST-iDTR mice with or without DT (non-DT) injection were subcutaneously transplanted into wild-type (WT) recipient mice. Tumor incidence, described as transplantation sites with tumors/transplanted mice amount (ratio), was measured at 18 weeks after transplantation. B PCR arrays were used to compare the expression of oncogenes and tumor suppressor genes between malignant AT2 and non-malignant AT2 groups. The red and green colors represent, respectively, increased or decreased gene expression in the heatmap for the malignant and non-malignant AT2 groups. Myc located at F07 (Extended Data Fig. 6A). C qRT-PCR analysis for Myc expression in the indicated cells. E-MDC and M-MDC cells were GFP-positive cells FACS sorted from the lungs of 4-week-old (E-MDC, n = 15) or 16-week-old (M-MDC, n = 3) MPG-ST-iDTR mice (GFP-positive cells FACS sorted from the mammary ADH tissue of 4-week-old MPG-ST-iDTR mice were used as control); N-AT2 and M-AT2 cells were tdTomato-positive cells FACS sorted from the parenchyma (N-AT2, n = 3) or lung metastasis (M-AT2, n = 3) of the MPG-ST-iDTR mice 2 weeks after DT treatment (tdTomato-positive cells FACS sorted from the peritumoral tissues of MPG-ST-iDTR mice before DT treatment were used as the control). n = number of mice. Data presented are the mean of triplicate experiments. Error bars indicate standard deviation (**P < 0.01; N.S, No significance). D Immunoblotting for anti-Myc in E-MDCs, M-MDCs, N-AT2 cells, M-AT2 cells (as described in C and their secreted exosomes (a). Quantification of Myc expression level (b). GAPDH was used as a control. Data presented are the mean of triplicate experiments. Error bars indicate standard deviation. E RNA expression of H19 lncRNA in the indicated cells (as described in C) and their secreted exosomes were analyzed by qRT-PCR and normalized to U6 snRNA. Data presented are the mean of triplicate experiments. Error bars indicate standard deviation. F and G FACS-sorted tdTomato-positive cells from the lung of 4-week-old ST mice, MPG-ST mice, or MPG-ST-iDTR mice were co-cultured with M-MDCs (as described in C) or M-MDCs with exosome inhibitor GW4869 (F, without M-MDCs as control) or with just exosomes collected from M-MDCs (G, without exosomes as control). The RNA level of H19 LncRNA was measured by qRT-PCR and normalized to U6 snRNA. Data presented are the mean of triplicate experiments. Error bars indicate standard deviation (**P < 0.01; *P < 0.05). Abbreviations: MPG, MMTV–PyMT-Green; MGP-ST, MPG/Sftpc-Tomato; MPG-ST-iDTR, MGP/ST/Cre-inducible diphtheria toxin receptor; MDCs, mammary gland-derived cells; E-MDCs, early MDCs; M-MDCs, malignant MDCs; N-AT2, non-malignant AT2; M-AT2, malignant AT2; C, control