Figure 1.

Synergistically enhanced lytic effects of combinations of cytotoxins and PLA₂s from the same and different species. (A) Low lytic concentrations of cytotoxins (Nn18: 8 µg/ml, Nm17: 6 µg/ml, Nn20: 20 µg/ml, Nmo9: 1 µg/ml, and melittin: 5 µg/ml) were mixed with sublytic concentrations of PLA₂s (Nmo12: 50 µg/ml, bvPLA₂: 50 µg/ml, and MII: 12 µg/ml) and the combinations were added to human keratinocytes (N/TERT). Controls were performed with N/TERT cells not subjected to the toxins. Synergistically enhanced cytotoxicity was examined after 24 h of incubation by determination of adenosine triphosphate (ATP) levels through a luminescent cell viability assay. Experiments were performed in triplicates with two replicates for each combination, and results are expressed as mean ± SD. Data was analyzed by an analysis of variance (ANOVA) test followed by a Bonferroni post-test. (*p < 0.001 compared to the respective effect with the individual cytotoxin). (B) Representative morphological features of N/TERT cells captured by Evo XL imaging system using a 4× objective lens. (a-c) Standard N/TERT cell cultures forming islands of adherent and flattened keratinocytes, indicating viable cells: a: control; b: bvPLA₂, and c: Nn18. (d-f) N/TERT cell cultures forming separated clusters of keratinocytes (fragmentation) and presenting several rounded cells, indicating cell damage and lysis: d: Nn18 + bvPLA₂; e: Nn18 + Nmo12, and f: Nn18 + MII. Similar patterns were seen for other toxins.