Figure 5

TUNEL assay to detect DNA fragmentation by confocal microscopy. Images are fluorescent confocal staining at ×600 magnification. Negative control was unchallenged HGECs at 24 h (A). Positive control was HGECs treated with DNase 1000 U/ml (B). HGECs were challenged with filtered supernatant of P. gingivalis 33277 culture (C) for 24 h. Additional plates (D to F) show challenge with live P. gingivalis 33277 supernatant pretreated with leupeptin, a selective Rgp inhibitor (D), zFKck, a selective Kgp inhibitor (E), or a cocktail of both inhibitors to inhibit total gingipain activity (F). Challenge for 24 hours with filtered culture supernatant derived from the RgpA/RgpB mutant E8 (G), the Kgp mutant K1A (H) or the RgpA/RgpB/Kgp mutant KDP128 (I), are also shown.