Fig. 3
Linc00210 was required for liver TIC self-renewal. a Impaired self-renewal of linc00210 silenced cells. Linc00210 were silenced with antisense oligo (ASO) (left panels), followed by sphere formation assays. Representative sphere images were shown in middle panels and calculated ratios were shown in right panels. For ASO transfection, 1 × 105 primary cells were transfected with 0.7 μL jetPEI-Hepatocyte reagent (MBTR005, Himedia Company) containing 1 μg ASO. The transfection reagent was removed 24 h later and knockdown efficiency was examined 48 h later, followed by sphere formation. b Primary cells were treated with ASO and CD133 expression levels were examined by realtime PCR. Three samples were used and the mRNA levels were normalized to control cells. c Sequential sphere formation assays were performed using linc00210 silenced and control spheres. Three samples were used. dLinc00210 silenced and control cells were used for invasive capacity. Typical images and cell numbers (mean ± s.d.) were shown. e 1 × 106 indicated cells were subcutaneously injected into BALB/c nude mice, and the established tumors were obtained one month later. Six mice were used for tumor propagation. f, g Tumor initiating capacities of the indicated cells were examined using gradient dilution xenograft model. 10, 1 × 102, 1 × 103, 1 × 104, and 1 × 105linc00210 silenced cells and control cells were subcutaneously injected into 6-week-old BALB/c nude mice. Tumor formation was observed 3 months later and the ratios of tumor-free mice were calculated (f). TIC ratios were calculated through extreme limiting dilution analysis (g). CI, Confidence interval; vs, versus. hLinc00210 was transcriptionally repressed through CRISPRi strategy (left panels), followed by sphere formation. Typical images (middle panels) and calculated ratios (right panels) were shown. i Lnc00210 depleted cells were established through CRISPRi strategy and CD133 expression levels were examined by realtime PCR. Three samples were used and the mRNA levels were normalized to control cells. j 1 × 106linc00210 silenced cells were subcutaneously injected into BALB/c nude mice, and the established tumors were obtained one month later. Six mice were used for tumor propagation. kLinc00210 overexpressed cells were established (left panels), followed by sphere formation assay (middle and right panels). l CD133 expression levels in lin00210 overexpressed and control cells were examined by realtime PCR. m Tumor propagation of linc00210 overexpressed and control cells. 1 × 106 indicated cells were used per mouse, and six mice were used for each group. n, o 10, 1 × 102, 1 × 103, 1 × 104, and 1 × 105linc00210 overexpressing cells and control cells were subcutaneously injected into 6-week-old BALB/c nude mice for tumor initiation. The ratios of tumor-free mice (n) and tumor initiating cells (o) were shown. CI, Confidence interval; vs, versus. For A, H, K, scale bars, 500 μm. Data were shown as means±s.d. **P < 0.01; ***P < 0.001 by two-tailed Student’s t test. Data are representative of three independent experiments