Fig. 5

Functional role of nigritoxin domains. a Cytotoxic activity of wild type (domains schematically represented in blue: N-terminal; yellow: central; magenta: C-terminal), truncated forms, and mutants (Δrabbit ears: deletion of the protruding loops; R,W->A,A: Arg512 and Trp540 replaced by two Ala; H, H->A, A: His598 and His650 replaced by two Ala) of nigritoxin on insect cells. Sf9 cells were incubated with 1.2 μM of truncated forms or mutants of nigritoxin or with the same volume of protein suspension buffer for 12 h. The cytotoxicity was monitored using Alamar Blue assay. For each condition, the cell viability was compared to mock (untreated) cells set at 100% viability and expressed in percentage (y-axis). The experiment was performed twice in triplicate and data are presented as mean ± S.D. Means with the same letter are not significantly different from each other (Kruskal–Wallis, p < 0.05). b Cellular localization of wild type, truncated, and mutant versions of nigritoxin in Sf9 cells. Cells were incubated with purified proteins/fragments (1.2 μM) for 1 and 12 h, fixed and detected by immunochemistry. Nuclei are stained in blue with DAPI. White arrows indicate nuclei damages. Scale bars: 20 μM