Fig. 2.
STAT3 knockdown with siRNA leads to NFκB activation in human melanoma cells. a–b Representative pictures of WM239 melanoma cells 48 h after transfection with siRNA labeled with Rhodamine (siGlo) using AMAXA electroporation. Using fluorescence microscopy (a) and flow cytometry analysis (b) the efficacy of siRNA transfection was determined as 84 %. c Effective STAT3 knockdown in WM239 cells. The STAT3 mRNA level was determined using qPCR and was related to its level in control siRNA transfected cells. d The levels of phosphorylated and total STAT3 and IκB proteins in cells transfected with control or STAT3 specific siRNA were examined by Western blotting. Immunoblots were re-probed with an antibody recognizing β-actin to ensure equal loading. Similar results were obtained in three independent experiments. The right panel shows quantification of the Western blots from three experiments using Image J with β-actin as the loading control. e The increase of the NFκB transcriptional activity in melanoma cells transfected with STAT3 specific siRNA. Cells growing onto 24-wells plates were co-transfected with the NFκB-luc plasmid and control or STAT3 siRNA using AMAXA electroporation. The luciferase activity was measured 48 h after transfection. The bars indicate mean values of luciferase activity in the mock transfected cells and cells transfected with the control or STAT3 siRNA. Data are presented as mean ± S.D. from three experiments, each in duplicate. f Evaluation of NFκB DNA binding by ELISA. Cells (1 × 107 cells/per group) were mock transfected or were co-transfected with a control or STAT3 siRNA using AMAXA electroporation. Cell nuclei extracts were collected 48 h after transfection and 2 μg of nuclear extract was subjected to an NFκB DNA binding assay (Active Motif—TransAM® NFκB Family Kit, Carlsbad, USA). The NFκB -ELISA assay results demonstrated an increase in the NFκB binding to DNA after silencing the expression of STAT3. Data are presented as mean ± S.D. from three experiments. g–h. STAT3 knockdown with siRNA induces specifically NFκB-Luciferase activity in WM209 and T1 melanoma cell lines. The NFκB-Luciferase activity measured in WM209 and T1 cells untreated (mock), treated with electroporation only or in cells transfected with two empty plasmids such as p-Super and PCMV6-XL5, or plasmids coding for shRNAs or siRNAs. An increase of the NFκB transcriptional activity was observed in melanoma cells transfected with plasmids encoding shRNA against STAT3 and control shRNAs and siRNA against STAT3 and control. Electroporation itself or transfection with empty plasmids do not induce NFκB activation. Data are presented as mean ± S.D. from three experiments