Briefly, cells at a density of 5 102 cells/well were plated in 96-well plates immediately and then infected with indicated adenoviruses or co-incubated with GSK-3 inhibitor CT99021 (3 M) for indicated duration. with Lv-lnc-p21 or Lv-ctrl (10 MOI). The diameter (C) and numbers of colonospheres (D) originating from single ALDH+ cells were measured with ImageJ software on Day 14. E. The numbers of soft agar colonies created by these cells were counted and shown as means SD. Colonies with a diameter higher than 75 m were counted. F. Stem cell markers, EpCAM, CD44, Lgr5, Nanog and Oct4, and differentiation markers Muc2 and CK-20 were examined by immunoblot analysis. Tubulin was a loading control. Representative graphs (B) or images (C, E, F) are shown. Data are offered as the mean SD (A, C, D, E) of each group from triple replicates. *< 0.05, **< 0.01. We constructed a lentiviral vector transporting lincRNA-p21 (Lv-lnc-p21) to restore its expression in ALDH+ CSCs to the levels that were comparable to ALDH? counterparts (Supplementary Physique S1D). Amazingly, the pool of ALDH+ cells was greatly reduced by Lv-lnc-p21 contamination (Physique ?(Figure1B).1B). Analysis of tumorsphere formation, a hallmark Akt1 and Akt2-IN-1 of CSCs [6, 22], indicated that Lv-lnc-p21-infected ALDH+ CSCs created smaller (Physique ?(Figure1C)1C) and less (Figure ?(Figure1D)1D) tumorspheres than those infected with control lentiviral vector (Lv-ctrl). Moreover, Lv-lnc-p21 infection reduced the growth of ALDH+ CSCs (Supplementary Physique S1E). Importantly, soft agar colony formation assays exhibited that expression of exogenous lincRNA-p21 suppressed the tumorigenicity of single ALDH+ CSCs (Physique ?(Figure1E1E). Furthermore, we examined the expression levels of other putative CRC CSC markers, such as EpCAM [23], CD44 [22], and Lgr5 [24], pluripotency factors Nanog and Oct4, and differentiation markers of colorectal epithelium, Mucin2 and CK-20. We found that lincRNA-p21 partially inhibited the expression of stemness-associated markers while upregulated the levels of differentiation-associated genes (Physique ?(Figure1F).1F). Taken together, these data demonstrate that exogenous lincRNA-p21 significantly inhibits CSC function and tumorigenicity and induces partial differentiation of CRC CSC, suggesting ER81 the possibility of restoring lincRNA-p21 to eliminate CRC CSCs. Depletion of lincRNA-p21 confers on ALDH? non-CSCs with stemness and tumorigenicity To further evaluate the role of lincRNA-p21 in the maintenance of CSC stemness, we employed lentiviral vectors that expressed two impartial shRNAs targeting lincRNA-p21 (Sh-lnc-p21a and Sh-lnc-p21b) to knockdown endogenous lincRNA-p21 in ALDH? CRC cells (Physique ?(Figure2A).2A). Interestingly, FACS analysis revealed that this ALDH? cells were in part transformed to ALDH+ ones by Sh-lnc-p21-contamination (Physique ?(Physique2B),2B), while these changes were not observed in Sh-GFP-infected cells, implying that loss of lincRNA-p21 may induce de-differentiation of ALDH? cells to generate ALDH+ CSCs. Open in a separate windows Physique 2 Knockdown of lincRNA-p21 enhances stemness and tumorigenicity of ALDHCCRC cellsA. The level of lincRNA-p21 was evaluated by qPCR in ALDH? cells infected with lentivirus expressing two impartial lincRNA shRNAs, Sh-lnc-p21a and Sh-lnc-p21b, at 10 MOI for 48 hrs. Contamination with shRNA targeting GFP, Sh-GFP, served as control. B. Circulation cytometric analysis of ALDH+ populace in ALDH? cells Akt1 and Akt2-IN-1 after transduction for 7 days. Akt1 and Akt2-IN-1 C, D. The diameter (C) and numbers of spheres (D) generating by single ALDH? cells were measured with ImageJ software 14 days after contamination with lentiviruses. More than 10 repeat wells were counted for each group and spheres with a diameter larger than 50 m were included. E. Numbers of colonies created Akt1 and Akt2-IN-1 by ALDH? cells with or without lincRNA-p21 knockdown in soft agar-containing medium. Colonies with a diameter higher than 75 m were.