TUNEL assay to detect DNA fragmentation by confocal microscopy. Images are fluorescent confocal staining at ×600 magnification. Negative control was unchallenged HGECs (A). Positive control was HGECs treated with DNase 1000 U/ml (B). HGECs were challenged with live (C) and heat-killed (D) P. gingivalis 33277 MOI:100 for 24 h. Challenge with MOI:100 for 4 h and MOI:10 for 4 and 24 h gave no staining (data not shown). Additional plates (E to G) show challenge with live P. gingivalis 33277 at MOI:100 for 24 h that were pretreated with leupeptin, a selective Rgp inhibitor (E), zFKck, a selective Kgp inhibitor (F), or a cocktail of both inhibitors to inhibit total gingipain activity (G). Challenge with P. gingivalis W50 (H), the RgpA/RgpB mutant E8 (I), the Kgp mutant K1A (J) or the RgpA/RgpB/Kgp mutant KDP128 (K), at MOI:100 for 24 h are also shown.