Fig. 4

Targeting STAT3 with its specific inhibitors reduced the formation and survival of HT29-derived tumorspheres. (a) Inhibitors against STAT3 were selected because RNAseq analysis/NetworkAnalyst indicated STAT3 overexpression in the tumorspheres. First, the compounds were added to HCT116 and HT29 cells individually in a dose-dependent manner and incubated for 48 h. (b) The IC50 of each compound revealed that napabucasin inhibited HCT116 cells and homoharringtonine specifically inhibited HT29 cells. (c) Napabucasin and homoharringtonine were consequently investigated for their activities against the formation and survival of the HT29-derived tumorspheres by using 0.1 and 0.5 μM of compound and incubating for 7 days. The results indicated that napabucasin and homoharringtonine both significantly reduced the formation and survival of HT29CSCs; however, homoharringtonine priorly inhibited the HT29-derived tumorspheres. (d) To investigate the growth factors influencing STAT3 activation, Western blot analysis and qPCR were used for observing the STAT3 phosphorylation and stem cell marker LGR5 levels. The results demonstrated that EGF played a major role in activating STAT3 and increasing LGR5 levels. *p < 0.05. ***p < 0.001