FIGURE 4.

Effects of IRF6 knockdown on PMA-inducible keratinocyte differentiation. A–L, OKF6 cells (A–I)and (J–L) human epidermal keratinocytes were transfected with IRF6-targeting (black columns) or control non-targeting (white columns) siRNA. A–I, 48 h post-transfection (A), IRF6 mRNA levels were measured by real-time PCR. IRF6 mRNA levels in cells transfected with the control non-targeting siRNA were given an arbitrary value of 100%. **, p < 0.01. B and C, OKF6 cells were treated with 100 ng/ml PMA or 0.1% DMSO vehicle for 24 h. IVL (B) and KRT13 (C) mRNA levels were measured by real-time PCR and are shown as the fold increase relative to DMSO-treated cells. *, p < 0.05. D–F, OKF6 cells were treated with 100 ng/ml PMA or 0.1% DMSO for 48 h and (D) viable cell number then enumerated by cell counting. *, p < 0.05. E, cells were lysed and subjected to Western blotting with anti-PCNA and anti-ERK2 (loading control) antibodies. F, PCNA protein levels in cells transfected with the control non-targeting siRNA and subsequently treated with DMSO as in E were given an arbitrary value of 100%. *, p < 0.05. G–I, OKF6 cells were treated with 100 ng/ml PMA or 0.1% DMSO for 2 h (OVOL1), 6 h (GRHL3), and 24 h (MAD1). GRHL3 (G), OVOL1 (H), and MAD1 (I) mRNA levels were measured by real-time PCR and are shown as the fold increase relative to DMSO-treated cells. *, p < 0.05. J–L, 72 h post-transfection, epidermal keratinocytes were treated with 100 ng/ml PMA or 0.1% DMSO for 6 h (GRHL3 and OVOL1) and 24 h (IVL). GRHL3 (J), OVOL1 (K), and IVL (L) mRNA levels were measured by real-time PCR and are shown as the fold increase relative to DMSO-treated cells. *, p < 0.05; **, p < 0.01. All graphical data are combined from at least three independent experiments.