Fig. 3

mTORC1 upregulates miR-130b-3p through activation of STAT3. A Tsc2 − / − or Tsc1 − / − MEFs were treated with 50 μM S3I-201 or DMSO for 24 h. B Tsc2 − / − MEFs or Tsc1 − / − MEFs were infected with lentiviruses encoding STAT3 shRNA (shSTAT3) or the control shRNA (shSc). C Tsc2 + / + MEFs were transduced with the retroviruses for STAT3C in pBabe or its control vector pBabe. A-C Cell lysates were subjected to western blot analysis using the indicated antibodies (up panels). The level of miR-130b-3p was detected by qRT-PCR (low panels). D and E Schematic illustration of the promoter region of miR-130b (D) and the two conserved STAT3-binding sites predicted by the JASPAR database (E). F The enrichment of H3K4me3 and STAT3 in the promoter of miR-130b was analyzed by ChIP-PCR assay. G Schematic illustration of the construction of miR-130b promoter luciferase reporters containing a region around site #1 with the STAT3 binding site intact (WT) or mutated (Mut). H HEK 293 T cells were co-transfected with WT-Luc or Mut-Luc plus pBabe-STAT3C or control pBabe and the internal control plasmid pRL-TK. Relative luciferase activity was detected 24 h after transfection. I Tsc2 + / + , Tsc2 − / − , and rapamycin (20 nM, 24 h) treated Tsc2 − / − MEFs were subjected to ChIP analysis with antibodies to p-STAT3 or control rabbit IgG. qRT-PCR was performed to amplify regions surrounding the putative STAT3 binding region (PBR) and a nonspecific STAT3 binding region (NBR). The data were plotted as the ratio of immunoprecipitated DNA to total input DNA. Error bars indicate mean ± SD of triplicate samples. **P < 0.01; *** P < 0.001; **** P < 0.0001