Fig. 5.
NANOG is a key downstream effector of interleukin (IL)-6/signal transducer and activator of transcription 3 (STAT3)/FRA1-driven CSC properties. a DLD1 cells were transfected with non-target control (siNC) or STAT3 small interfering RNAs (siRNAs) and incubated with IL-6 for the indicated times. The protein levels of STAT3-pY705, STAT3, FRA1, NANOG, and GAPDH was examined by western blot analysis with the respective specific antibodies. b Parental (FOSL1+/+) and FOSL1−/− HT-29 cells were cultured in the presence/absence of IL-6 for 24 h. The protein levels of STAT3-pY705, STAT3, FRA1, NANOG, and GAPDH was examined by western blot analysis with the respective specific antibodies. c–e Chemo-resistance (c), sphere (d), and xenograft (e) formation assays of DLD1 cells featuring different combinations of FOSL1 knockdown, NANOG overexpression, and IL-6 stimulation (shNC = control for the siRNA-driven FOSL1 knockdown). f, g Migration and invasion (f) and sphere formation (g) assays were performed with shScramble (control for the shRNA-driven NANOG knockdown) and shNANOG DLD1 cells cultured in the presence/absence of IL-6. Results are shown as histograms showing quantitative values of the number of cells (or spheres) from triplicate experiments (mean ± SD). h shScramble and shNANOG DLD1 cells were cultured in medium supplemented with the chemotherapeutic drugs 5-Fluorouracil and Cisplatin and in the presence/absence of IL-6 (50 ng/ml). The percentage of apoptotic (Annexin V+) cells indicate that shNANOG DLD1 cells were characterized by increased sensitivity when compared to the shScramble cells. IL-6 enhances chemo-resistance in shNC DLD1 cells, an effect that is abrogated by the NANOG knockdown. i 5 × 105 shScramble and shNANOG DLD1 cells were cultured in the presence/absence of IL-6 for 5 days and then injected subcutaneously into the flanks of BALB/C nude mice. In the shScramble group, DLD1 cells treated with IL-6 formed larger tumors, an effect that was abolished upon NANOG knockdown. *p < 0.05, **p < 0.01, ***p < 0.001. Unpaired t test. Data are presented as mean ± SD