Fig. 3

STAT3 trans-activates miR-181b transcription. a Western blot analysis of p-STAT3 and STAT3 in Eca109 parental cells and tumor sphere cells. b, c Colony formation assays analysis of tumor sphere cells with or without STAT3. Colonies were counted following 0.5 % crystal violet staining. Triplicate independent experiments were performed. Scale bar = 10 mm. d qPCR analysis of miR-181b expression in SFCs treated with JSI-124 and with or without STAT3. e qPCR analysis of miR-181b expression levels in SFCs treated with IL-6. f Bioinformatics analysis of predicted binding sites for STAT3 at the promoter of miR-181b. Schematic representation of the 1650-bp regulatory region upstream of the human miR-181b-stem-loop. The E-box motifs were predicted at-1650 bp relative to the transcription start site of the human miR-181b stem-loop. g Luciferase assays for promoter activity. SFCs were dissociated with trypsin and cotransfected with different promoter constructs (wild-type or mutant) and vector expressing STAT3. h qPCR analysis for expression levels of mature miR-181b, pri-miR-181b in SFCs treated with vector expressing STAT3. β-Actin and U6 snRNA served as internal controls. SFCs, sphere formation cells. Error bars represent mean ± SD