Fig. 2.

CircEPHB4 necessarily and sufficiently promoted cancer stemness of glioma cells. (A,B) The expression levels of circEPHB4 were measured by qRT‐PCR and compared between indicated cells. (C,D) The stemness of indicated cells was examined by neurosphere formation assay, with representative images of formed neurospheres shown in (C) and the quantification results shown in (D). (E,F) The expression of CD133 on the surface of indicated cells was examined by flow cytometry in (E) and the quantification results are shown in (F). (G,H) The expression levels of stem‐cell markers Nestin, Oct4, Nanog, CD133 and CD44 were measured by Western blotting in indicated cells (G) and quantified as the ratio to GAPDH (H). LN229 or SHG‐44 cells were engineered either to stably knock down (shcircEPHB4) or to overexpress circEPHB4 (OE‐circEPHB4). shNC or OE‐NC vector was used as the corresponding negative control for shcircEPHB4 or OE‐circEPHB4. Data are presented as mean ± SD from three independent experiments. Comparison between two groups was performed using Student’s t‐test and between three or more groups using one‐way ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01.