Fig. 2

CircYap inhibited the translation of Yap protein. a Polysome of the vector control and circYap overexpressed cells were extracted and subjected to 10 to 50% sucrose gradient by ultracentrifuge. Twenty-four polysome fractions were collected from top to bottom followed by RNA extraction. Yap (right), mdm2 (middle) and GAPDH (left) mRNA expression in each fraction were determined by real-time PCR (upper) and visualized by DNA agarose gel (lower). n = 3. b Left, the cytosolic and nuclear expression of circYap in immortalized non-cancerous cell and tumor cell lines were examined by real-time PCR. The relative ratio of cytosolic to nuclear expression was calculated in different cells respectively. n = 6. **p < 0.01 compared to the cytosolic/nuclear ration of circYap in HGF cells. Right, the cytosolic and nuclear expression of circYap and Yap mRNA in HepG2 cells were determined. The expression of cytosolic and nuclear GAPDH and U6 were used to examine the purity of cytosolic and nuclear RNA. n = 6. **p < 0.01 compared to nuclear expression of each RNA. c The cytosolic and nuclear expression of circYap (left) and Yap mRNA (right) in vector control and circYap overexpressed cells were determined. n = 6. **p < 0.01 compared to vector control. (d) The cytosolic and nuclear levels of Yap protein in vector- and circYap-transfected MDA-MB231cells were examined by Western blotting. Tubulin and PCNA antibodies were used to examine the purity of cytosolic and nuclear fractions, respectively. e Full length linear Yap mRNA was knocked down by Yap siRNA (si-Yap1 and si-Yap2) in MDA-MB231 cells. Yap protein expression was examined by Western blotting in wide type (wt), siRNA control (si-ctrl), si-Yap1 or si-Yap2. GAPDH protein was examined as loading control. f Upper, circYap was knockdown by circYap siRNAs (si-cirYap1 and si-cirYap2) in MDA-MB231 cells. The expression of circYap and Yap mRNA were examined. n = 6. **p < 0.01 compared to si-ctrl. Lower, Yap protein levels were examined by Western blotting. g General translation rates were examined by cap pull-down assay in wide type (wt), vector, circYap or its linear precursor (linYap), or full-length Yap mRNA overexpressed (Yap) cells. The m7GTP bound eIF4G and eIF4E were detected along with their input in total cell lysate