Figure 3. ZIC2 deficiency abrogates self-renewal of liver CSCs and tumor propagation.

(A) ZIC2-deficient cells were established using a CRISPR/Cas9 system. T7 endonuclease I cleavage confirmed the efficiency of sgRNA (white arrowheads), and ZIC2 KO efficiency was detected by Western blot. ZIC2 was deleted in 8 cell lines. (B) ZIC2 deficiency impairs self-renewal of HCC cells. ZIC2 KO cells and control cells were cultured for sphere formation. (C) ZIC2-deficient and control Hep3B cells (1 × 10⁶) were injected into BALB/c nude mice. Tumor sizes were measured every 5 days. (D) ZIC2 deficiency reduces tumor-initiating capacity. The indicated Hep3B cells were implanted into BALB/c nude mice for tumor initiation. Percentages of tumor-formation mice were calculated (left panel), and established tumors were shown (right panel). (E) ZIC2 was silenced in HCC primary tumor cells. (F) ZIC2-silenced cells (shZIC2) and scramble control cells (shCtrl) were cultured for sphere formation. Six HCC samples obtained similar observations. Z2KO, ZIC2 KO cell. Scale bars: 500 μm (B and F). Data are shown as means ± SD. Two-tailed Student’s t test was used for statistical analysis. *P < 0.05; **P < 0.01; and ***P < 0.001. Data are representative of at least 3 independent experiments.