Figure 7.

Heterogeneity of circSKA3 expression

(A) Single-cell colonies were obtained and counted after 3 weeks of culture. (B) At least 200 cells were collected and pooled into one sample (n = 1) from small colonies (containing less than 50 cells; n = 52), regular culture (Regul; n = 7), low-invasion colonies (LICs; n = 7), middle colonies (200–300 cells; n = 48), and large colonies (>500 cells; n = 167). All samples were processed to real-time PCR. LICs and small and middle colonies showed decreased circSKA3 levels. (C) LICs and small and middle colonies also showed decreased c-myc levels. (D) Levels of circSKA3 and c-myc in 167 large colonies were not correlated. (E) In a cell invasion assay, each Matrigel-coated chamber insert was loaded with 200 cells harvested from regular colonies, LICs, and small, middle, and large colonies (n = 4). All large colonies were grouped into circSKA3^H/c-myc^H, circSKA3^H/c-myc^L, circSKA3^L/c-myc^H, and circSKA3^L/c-myc^L. LIC and small- and middle-colony cells showed a decreased invasion ability, while circSKA3^H colonies showed an enhanced invasion. (F) In cell migration assays, large colonies with high levels of circSKA3, but not c-myc, promoted cell activities. (G) The large-colony cells (circSKA3^H/c-myc^H) were subjected to single-cell PCR to measure circSKA3 levels. A similar pattern of circSKA3 distribution was observed in the regular culture. ∗∗ p<0.01; Error bars, SD