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. 2019 Jan 24;9(3):811–828. doi: 10.7150/thno.29271

Figure 8.

Figure 8

Elevated Nanog expression plays an essential role in RACK1-regulated HCC CSCs. (A-G) 96 h after HuH7 cells were infected with the indicated lentivirus, cells were subjected to the following assays: (A) Immunoblotting analysis of the expression of Nanog and RACK1 in total cells. (B) Flow cytometric analysis of CD13 expression in total cells. (C) Immunoblotting analysis of the expression of Nanog and RACK1 in sorted CD13+ cells. (D) Sphere formation assays of sorted CD13+ subpopulation. mean±s.d. (n=3); *P<0.05, **P<0.01 (E) In vivo tumorigenicity experiments of CD13+ subpopulation (5000 cells/site, 7 weeks, n=6). (F) Etoposide (Etop, 100 μM, 48 h)- or sorafenib (Sora, 50 μM, 24 h)-induced apoptosis of CD13+ subpopulation. mean±s.d. (n=3). (G) qRT-PCR analysis of sorted CD13+ subpopulation for the expression of the indicated drug-resistant relative genes. mean±s.d. (n=3). (H-K) 24 h after HuH7 cells were transfected with mammalian expression vector encoding GFP-tagged wild type RACK1 or RACK1 mutant lacking WD5, GFP+CD13+ subpopulations were sorted and subjected to immunoblotting (H), sphere formation assays, mean±s.d. (n=3) (I), etoposide (Etop, 100 μM, 48 h)- or sorafenib (Sora, 50 μM, 24 h)-induced apoptosis, mean±s.d. (n=3) (J), and qRT-PCR analysis for the expression of the indicated drug-resistant relative genes. mean±s.d. (n=3) (K).