Figure 3.

Grainyhead-like 2 (GRHL2) is required for the junction protein expression and the maintenance of epithelial barrier function in oral epithelial cells. (A) GRHL2 was knocked down in HOK-16B cells by 2 independent Grhl2 ShRNAs (Sh1 and Sh2). Western blotting was performed to assess the level of GRHL2, claudin (CLDN)–1, and CLDN-4. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was probed for loading control. (B) In HOK-16B cells with GRHL2 knockdown, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to assess the level of GRHL2, β-catenin (β-cat), E-cadherin (E-cad), CLDN-1, CLDN-3, CLDN-4, occludin (OCLN), and zonula occludens (ZO)–1 messenger RNA (mRNA). (C) qRT-PCR was performed with total RNAs isolated from normal human keratinocyte (NHK)/GRHL2 or NHK/LXSN to assess the mRNA levels of junction molecules (β-cat; E-cad; CLDN-1, -3, -4; OCLN; and ZO-1). (D) Immunofluorescent staining was performed to examine GRHL2 and junction protein (E-cad, ZO-1, and CLDN-4) expression in HOK-16B cells with or without GRHL2 knockdown. (E) Permeability of FITC-dextran across confluent HOK-16B monolayer was determined using the cells exposed to Porphyromonas gingivalis (Pg) LPS (1 or 5 µg/mL) for 24 h or the cells with GRHL2 knockdown (ShGRHL2 vs. ShContr). Error bars indicate mean/SD. *Statistically significant (P < 0.05) compared with the control cells. (F) Permeability of Pg across HOK-16B cell monolayer with or without GRHL2 knockdown was determined at different time intervals after Pg bacterial inoculation using the transwell culture system. Pg bacterial content in the bottom chamber was determined by quantitative polymerase chain reaction using Pg-specific primer sets. Error bars indicate mean/SD. *Statistically significant (P < 0.05) compared with the control cells.