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. 2016 Jun 13;7:ncomms11840. doi: 10.1038/ncomms11840

Figure 4. The splicing switch to TEAD4-S inhibits EMT and cancer cell proliferation.

Figure 4

(a) Schematics of the engineered splicing factor (ESF-TEAD4) that regulates the splicing of TEAD4. PUF domain was engineered to recognize an 8-nt RNA sequence (GAGCCTTG) in exon 3 of TEAD4, and fused with a glycine-rich motif of hnRNP A1 that can inhibit exon inclusion on binding to pre-mRNA. The resulting ESF, ESF-TEAD4, was designed to bind and inhibit the inclusion of exon 3 in TEAD4, thus inducing splicing switch towards TEAD4-S. (b) ESF-TEAD4 or control ESF was transfected into YAP-expressing H157 and A549 cells. RNAs were extracted from the transfected cells, and the splicing of TEAD4 was examined with RT–PCR. The representative gel was shown. The mean±s.d. for the percentage of TEAD4-S mRNA was plotted from three experiments. (c) Total proteins were purified from the same set of transfected cells as in b, and the epithelial and mesenchymal markers were detected with western blots. (d) ESF-TEAD4 or control ESF was transfected into YAP-expressing H157 or A549 cells. Colony formation assay was carried out to examine tumour cell proliferation. All experiments were performed in triplicates, with mean±s.d. of relative colony numbers plotted (P values from t-test). Images of the whole plate were shown.