10.1371/journal.ppat.1008124.g008 Hiroki Takeuchi Hiroki Takeuchi Naoko Sasaki Naoko Sasaki Shunsuke Yamaga Shunsuke Yamaga Masae Kuboniwa Masae Kuboniwa Michiya Matsusaki Michiya Matsusaki Atsuo Amano Atsuo Amano <i>P</i>. <i>gingivalis</i> gingipains penetrate the epithelial barrier of IHGE cells. Public Library of Science 2019 Lys-specific cysteine proteases Gingival epithelial cells LPS degradation JAM 1 homodimers 40 kDa dextran gingipain gingival epithelial interface gingival epithelial cells JAM 1. Knockdown JAM 1 PGN junctional adhesion molecule junctional adhesion molecule 1 Porphyromonas gingivalis gingival epithelium epithelial barrier function 2019-11-07 18:59:56 Figure https://plos.figshare.com/articles/figure/_i_P_i_i_gingivalis_i_gingipains_penetrate_the_epithelial_barrier_of_IHGE_cells_/10269347 <p><b>(A, B)</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 <i>P</i>. <i>gingivalis</i> WT or the Δ<i>kgp</i> Δ<i>rgpA</i> Δ<i>rgpB</i> 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. <b>(C, D)</b> 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 <i>P</i>. <i>gingivalis</i> WT or the Δ<i>kgp</i> Δ<i>rgpA</i> Δ<i>rgpB</i> 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. <b>(E, F)</b> 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 <i>P</i>. <i>gingivalis</i> WT or the Δ<i>kgp</i> Δ<i>rgpA</i> Δ<i>rgpB</i> 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. <b>(G-I)</b> Schematic image of the culture insert system (G). JAM1-expressing IHGE cells (WT or overexpressing JAM) were infected with <i>P</i>. <i>gingivalis</i> 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 <i>P</i>. <i>gingivalis</i> 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.</p>