Fig. 2: Altered NF-κB activity in mesothelioma cell lines Ist-Mes2 and MPP89 compared to normal mesothelial cells HMC35.

A IST-Mes-2, MPP89, and HMC35 cells (3 × 10⁶) cells were stimulated or not with TNF-α. After 24 h, cells were co-transfected with pSV-β-Gal (0.2 µg) and pκBluc, (0.2 µg), then were followed for additional 48 h. The ratio of firefly luciferase activity to β-galactosidase activity was expressed as relative light units. Mean values ± SE of 3 independent experiments are shown. B The binding of p65, p50, p52, c-Rel, and RelB proteins to the NF-κB double-stranded oligonucleotide was measured in nuclear extracts using the NF-κB Combo Transcription Factor ELISA assay kit. Values are the mean ± SE of three independent experiments. C IST-Mes-2, MPP89, and HMC35 cells (3 × 10⁶) cells were stimulated or not with TNF-α for 24 h. p65 was detected by Western blotting of nuclear cell extracts (30 μg). Histone-H1 was included as control of protein loading. Phosphorylated form of IKK and total form of IKK were detected by Western blotting of cytosolic cell extracts (30 μg). Vinculin was included as control of protein loading. D Cells were stimulated or not with TNF-α for 24 h and the IKK activity was measured in cytosolic cell extracts (30 μg) using the HTScan IKK kinase assay kit. Values are the mean ± SE of three independent experiments. The asterisks indicate statistically significant differences between stimulated and unstimulated cells, according to Student’s t-test (p < 0.05).