Figure 4.

Colocalization of circSKA3 with Tks5 and Itgb1

(A) Left: RNAs extracted from nuclei, cytosol, and membrane were subjected to real-time PCR. GAPDH and linear SKA3 were mainly expressed in the cytosol, whereas circSKA3 was highly expressed in the cell membrane. Right: the membrane fraction had high levels of circSKA3 in circSKA3-overexpressing cells compared with those in WT and vector-transfected cells. ∗∗p < 0.01. Error bars, SD (n = 4). (B) MB-231 cells were cultured on gelatin-coated culture dish for 24 h. Subcellular fractions and total invadopodia were isolated for real-time PCR. High levels of circSKA3 were detected in the invadopodia and cell membrane (left). SKA3 mRNA was mainly detected in cytosol (right). ∗∗p < 0.01. Error bars, SD (n = 4). (C) MB-231 cells transfected with circSKA3 siRNAs or control oligos were cultured on gelatin-coated dishes for 24 h. Real-time PCR showed decreased levels of circSKA3 in the gelatin of siRNA-transfected cells. ∗∗p < 0.01. Error bars, SD (n = 4). (D) Silencing circSKA3 increased Tks5 levels in the cytosol, but decreased Tks5 in the cell membrane. (E) Overexpression of circSKA3 promoted Tks5 translocation from cytosol to cell membrane. (F) The membrane fraction of MB-231 showed higher levels of Tks5 than MCF-7 cells. (G) Cell lysates were subjected to immunoprecipitation, followed by real-time PCR with primers specific for circRNAs as shown. Antibodies against Tks5 and Itgb1 pulled down circSKA3, but not the others. ∗∗p < 0.01. Error bars, SD (n = 4).