Figure 2.

HotairM1 Is Required for the Self-Renewal Maintenance of CSCs

(A) HotairM1 was silenced in tumor cells by two independent shRNAs. HotairM1 silencing efficiency was determined using quantitative real-time PCR. Data are indicated as the mean ± SD. (B) Pluripotent transcription factors (Oct4, Sox2, and Nanog) were analyzed in HotairM1-depleted cells using western blot. (C) Onco-sphere formation assay using serum-free medium was performed to detect the sphere-forming capacity. Representative images of tumor spheres at a dose of 1,000 cells per well are shown. (D) Statistical analysis of spheres per 1,000 cells was performed. Data are indicated as the mean ± SD. (E) In vitro limiting dilution assay using different numbers of cells was performed. Spheres formed from different doses of cells were statistically analyzed. (F) HotairM1-silenced or shCtrl tumor cells were subcutaneously injected into BALB/c nude mice for observation of tumor growth. The results are shown as the mean ± SD. (G) HotairM1-silenced tumor cells and shCtrl cells were injected into mice in gradient doses of cells. The number of tumors formed in each mouse was calculated and analyzed. (H) Tumorigenic cell frequency in HotairM1-silenced and shCtrl tumor cells was analyzed with a limiting dilution assay (http://bioinf.wehi.edu.au/software/elda/). CI, confidence interval. ∗p < 0.05; ∗∗p < 0.01, by two-tailed Student’s t test.