Fig. 6

DUB activity of OTUD7B is required for inhibition of LCL161-induced cell elongation, invasion and migration. a A C194S/H358R(CH) mutant in the OTU domain was produced using the HA-OTUD7B plasmid. b H1299 cells were transfected with pcDNA-HA-OTUD7B(WT) or pcDNA-HA-OTUD7B(CH). After treatment with 10 μM LCL161 for 24 h, the cells were analysed for expression of TRAF3, NIK, p100, p52, and RelB by Western blotting. β-Actin expression served as a loading control. c H1299 cells were transfected with Flag-TRAF3 and HA-OTUD7B (WT) or Flag-OTUD7B (CH). After treatment with the indicated concentrations of LCL161, a ubiquitin assay in H1299 cells was performed by Western blotting using an anti-ubiquitin antibody. d-g H1299 cells were transfected with Flag-TRAF3 and HA-OTUD7B or Flag- OTUD7B(CH) and treated with the indicated concentrations of LCL161 for 48 h. d mRNA levels of NF-κB target genes were examined by quantitative RT-PCR. e Cell elongation was quantified by calculating the cell elongation index (length/width); the fold increase in the elongation index is shown. f The wound-healing process was monitored by an inverted light microscope. g Migrated cells were placed in a collagen-coated transwell migration chamber, fixed and stained with crystal violet, and optical density was measured. Representative results are shown. Plots are the mean ± SEM of data from three independent experiments. *P < 0.05