Diindolylmethane (DIM) inhibits signal transducer and activator of transcription 3 (STAT3) pathway in ovarian cancer cells. (A) Representative blots showing the concentration-dependent effect of DIM on phosphorylated (p)-STAT3 (Tyr-705), p-STAT3 (Ser-727), STAT3, Mcl-1, survivin in (i) SKOV-3, (ii) OVCAR-3, (iii) TOV-21G and (iv) A2780 ovarian cancer cells. Concentration-dependent effect of DIM on p-STAT3 (Tyr-705) and STAT3 in (v) OVCAR-429 and (vi) OVCAR-433 cells. Actin was used as loading control. (B) Effect of DIM on nuclear translocation of p-STAT3 and STAT3. Representative blots of nuclear lysates of (i) SKOV-3, (ii) OVCAR-3, (iii) TOV21G and (iv) A2780 cells treated with DIM. Lamin B was used as loading control. (C) Effect of DIM on STAT3 DNA binding. (i) SKOV-3 cells or (ii) OVCAR-3 cells were treated for 24 h with or without 75 μM DIM and nuclear cell extracts were tested for STAT3 DNA-binding activity as measured by the Universal EZ-TFA transcription factor colorimetric assay. (D) Effect of DIM on STAT3-regulated luciferase reporter activity. STAT3 luciferase transcriptional activity was determined in (i) SKOV-3, (ii) OVCAR-3, (iii) TOV21G or (iv) A2780 cells. Firefly luciferase activities were corrected for Renilla luciferase levels and then normalized relative to the control, which was considered as 100%. Means and SD of two independent experiments performed in triplicate are shown. The Student's t test was used for statistical analysis to compare control and DIM treatment.