P. gingivalis gingipains penetrate the epithelial barrier of IHGE cells.

(A, B) Schematic image of the culture insert system (A). A monolayer of IHGE cells was cultured in the upper compartment and on a coverslip in the lower compartment. Cells in the upper compartment were infected with P. gingivalis WT or the Δkgp ΔrgpA ΔrgpB mutant at an MOI of 100. Following 1 h of incubation, cells in the lower compartment were fixed, stained with DAPI (cyan) and anti-JAM1 (yellow), and analyzed by confocal microscopy (B). Scale bars, 10 μm. (C, D) Schematic image of the culture insert system (C). A monolayer of IHGE cells (WT) was cultured in the upper compartment and IHGE cells stably expressing Myc-mCherry–tagged HA-inserted JAM1 were cultured on a coverslip in the lower compartment. Cells in the upper compartment were infected with P. gingivalis WT or the Δkgp ΔrgpA ΔrgpB mutant at an MOI of 100. Following 1 h of incubation, cells in the lower compartment were fixed, stained with anti-HA (green), and analyzed by confocal microscopy (D). Scale bars, 10 μm. (E, F) Schematic image of the culture insert system (E). A monolayer of IHGE cells (WT) was cultured in the upper compartment and IHGE cells stably expressing HA-JAM1 Δ (1–133) or Δ (1–133) K134H R234H were cultured on a coverslip in the lower compartment. Cells in the upper compartment were infected with P. gingivalis WT or the Δkgp ΔrgpA ΔrgpB mutant at an MOI of 100. Following 1 h of incubation, cells in the lower compartment were fixed, stained with DAPI (cyan) and anti-HA (yellow), and analyzed by confocal microscopy (F). Scale bars, 10 μm. (G-I) Schematic image of the culture insert system (G). JAM1-expressing IHGE cells (WT or overexpressing JAM) were infected with P. gingivalis for 1 h and analyzed by immunoblotting with the indicated antibodies (H). A monolayer of IHGE cells (WT or overexpressing JAM1) was cultured in the culture insert. Cells were infected with P. gingivalis at an MOI of 100 in the upper compartment. Following 1 h of incubation, cells in the lower compartment were fixed, stained with DAPI (cyan) and anti-JAM1 (yellow), and analyzed by confocal microscopy (I). Scale bars, 10 μm.