Fig. 2
From: Circular RNA circIPO11 drives self-renewal of liver cancer initiating cells via Hedgehog signaling
CircIPO11 depletion impairs liver CSC self-renewal maintenance and inhibits HCC propagation. A, B CircIPO11 depletion decreased the capacity of oncosphere formation in HCC cells. Overexpression of circIPO11 (oecirc) rescued the sphere formation reduced by circIPO11 depletion (A). Scale bar, 500 μm. statistical results of sphere-formation ratios were tested by serial sphere formation assays (B). Data are shown as means ± SD. C Limited-diluted circIPO11 depletion or control Huh7 CSCs were subcutaneously injected into BALB/c nude mice for 3 months to detect tumor-bearing rates of mice. Tumor-free mice are counted (upper panel) and representative tumor images are shown (lower panel). n = 8 for each group. D 1 × 106 circIPO11-depleted or control HCC cells were subcutaneously injected into BALB/c nude mice. Two effective shRNAs were used. Representative tumor images are shown (right panel). Results are shown as means ± SD. n = 5 for each group. E Orthotopic liver tumor imaging of Huh7-Luc cells transduced with control or shcirc#1 or #2 vectors. Representative images are shown (left panel), and statistical results are shown as means ± SD (right panel). n = 5 for each group. F Representative images of clone formation capacity in circIPO11 depletion and control HCC cell lines (left panel), and statistical results are shown as means ± SD (right panel). G CircIPO11 expression levels in ASO-treated (40 ng/ml) Huh7 cells were detected followed by qRT-PCR. ASOs (#1, #2 and #3) were designed and two effective ASOs (#1 and #2) for corresponding experiments. ASOs were listed in Table S1. H ASOs (#1 and #2) against circIPO11 were used to treat HCC primary tumors in BALB/c nude mice. Twenty-five milligram per kilogram ASOs were injected around the tumor every 2 days (n = 5 mice per group). Scale bar, 1 cm. I CircIpo11 was completely abolished in circIpo11 KO mice by Northern blots. J Macroscopic tumor images of circIpo11+/+ and circIpo11−/− livers after treated by DEN for 8 months (8 M) and 12 months (12 M). Black arrows indicate liver tumors. n = 12 for each group. Scale bar, 1 cm. K Representative HE images of circIpo11+/+ and circIpo11−/− livers sections at 8 M and 12 M after DEN treatment. Scale bar, 100 μm. L, M Numbers of macroscopic tumors on liver surface (L), and liver-to-body weight ratios (M) were presented at 8 M and 12 M after DEN treatment. n = 12 for each group. *P < 0.05; **P < 0.01; *** P < 0.001 by two-tailed Student’s t-test. Data are representative of at least three independent experiments