Figure 7. Ad-lnc-p21-MRE suppresses the growth and stemness of CSCs in nude mice and exhibits limited toxicities to normal tissues.

A. ALDH⁺ HCT116 cells were infected with either Ad-EGFP, Ad-lnc-p21, or Ad-lnc-p21-MRE and then transplanted subcutaneously in a limiting dilution manner in nude mice. The frequencies of cancer-initiating cells (CICs) were shown. Secondary tumor formation assays were performed to examine self-renewal potential in vivo. See also Supplementary Figure 7A. B, C. ALDH⁺ HCT116 cells (5 × 10⁵) was inoculated into the flanks of each male BALB/c nude mice (4 group; n = 6). The tumor-bearing mice were treated with ether Ad-EGFP, Ad-lnc-p21, or Ad-lnc-p21-MRE via tail vein injection. Control mice were treated with PBS. Growth curves (B) and weights (C) of the xenograft tumors were shown. D, E. Xenograft tumor cells were dissociated to make single cell suspension. The percentages of ALDH⁺ cells were examined by flow cytometry (D) Colonosphere formation assay were performed using single xenograft tumor cells and the numbers of tumorspheres were shown (E) F. The xenograft tumor cells were collected to examine the expression of β-catenin in nuclear lysate and whole cell lysate (WCL) by western blot. Lamin B and GAPDH served as loading controls for nuclear lysate and WCL, respectively. G. Human lincRNA-p21 expression was detected in mouse liver by Northern blotting. U6 was used as control. H. Western blot analyses of β-catenin, Ki-67, and cleaved caspase-3 expression in the liver tissues of recipient mice. I. Blood ALT levels of tumor-free healthy mice that were administrated i.v. with the adenoviruses or PBS (4 group; n = 6). Representative graphs (D) or images (F, G, H) are shown. Data are presented as the mean ± SD (B, C, E, I) of each group from triple replicates. *P < 0.05, **P < 0.01.