Fig. 2

Mutation of the WR domain abrogates TWIST1 interaction with RELA. a Schematic representation of TWIST1 alleles used. Triple mutant contains W190A, R191A, and E193A mutations. b Co-IP reveals that single amino acid substitutions in the WR domain affect TWIST1-RELA binding, with the triple mutant producing a greater reduction in binding. c Quantitation of duplicate CoIP western blots. TWIST1 mutations lead to 50–60% reduction in RELA binding on average. Graphed is the ratio of TWIST1 to RELA, each normalized to its input for each condition. d Dual luciferase assay demonstrates that IL-8 promoter driven luciferase activity, a surrogate for IL-8 activation by the TWIST1-RELA complex, is influenced by TWIST1 mutation. As seen in the CoIP, single amino acid substitutions reduce FFluc expression by about 50% with respect to RELA alone, with the triple mutant producing a greater reduction. Graph represents firefly luciferase expression normalized to renilla luciferase for each condition. Error bars represent standard deviations of biological triplicate experiments. WT TWIST1 condition was used as the basis for statistical comparisons. pGL3 lacking the IL-8 promoter was used as a negative control. ***, p < .001; ****, p < .0001