Fig. 7

H19-S and Let-7a present in the same Ago2 complex in M-AT2 cells. A Schema for the alternative splicing of H19. B The online RNAhybrid tool predicted that the alternative splicing of H19 resulted in an emerging binding site for Let-7a. C Immunoprecipitation of Ago2 protein from cell lysates from M-MDC, I-AT2, and M-AT2 cells, with non-immune IgG control. The input lane indicates lysate samples used as a positive control for Ago2. D The tumor tissues (n = 13) of the lung metastasis were sorted for tdTomato-positive AT2 cells (I-AT2) or for GFP-positive MDC cells (M-MDC) from 16-week-old MPG-ST-iDTR mice without DT injection. The tdTomato-positive AT2 cells (M-AT2) were sorted from the lung metastasis (n = 5) of MPG-ST-iDTR mice two weeks after DT injection. qRT-PCR detection of H19-L or H19-S in M-MDC, I-AT2, and M-AT2 cells that were precipitated by Ago2 or negative control IgG and normalized to U6 snRNA. n = number of tumor tissues. Data presented are the mean of triplicate experiments. Error bars indicate standard deviation (**P < 0.01; *P < 0.05; N.S, No significance). E qRT-PCR detection of Let-7a endogenously associated with sponge RNA in M-MDC, immortal AT2 and M-AT2 cells that were pulled down by Ago2 or negative control IgG and normalized to U6 snRNA. Data presented are the mean of triplicate experiments. Error bars indicate standard deviation (**P < 0.01; N.S, No significance). F Scheme of Let-7a target luciferase reporter. G The luciferase activity assessment in normal AT2 cells transfected with sensor (psiCHECK2-let7a 4×), together with 0, 20, 40, 80, or 160 ng of sponge plasmid pH19-L, pH19-S or pH19-S mut. Data presented are the mean of triplicate experiments. Error bars indicate standard deviation (***P < 0.001; **P < 0.01; *P < 0.05; N.S, No significance). H Scheme of Myc pMirTarget luciferase reporter. I Activity of Myc pMirTarget luciferase reporter plasmids in N-AT2 cells co-transfected with Let-7a mimics or Let-7a mimics together with pH19-L, pH19-S, or pH19-S mut (normalized to control). C, control; M, mimics; S + M, pH19-S + mimics; L + M, pH19-L + mimics; SM + M, pH19-S mutant + mimics. Abbreviations: MDCs, mammary gland-derived cells; M-MDCs, malignant MDCs; M-AT2, malignant AT2 cells; I-AT2, immortalized AT2 cells