FIGURE 6.

(A) HaCaT and OKF6/TERT2 cells were transiently transfected with pCMV6, IRF6wt, and IRF6_VWS and stained with an anti-myc and anti-IRF6 antibody to analyze the localization of the exogenous proteins. Scale bar: 20 μm. Endogenous IRF6 was weakly detectable in both cell lines (pCMV6/anti-IRF6 panels) and is shown for HaCaT cells as a close-up (longer exposure). Scale bar: 10 μm. Note that IRF6_VWS was weaker expressed than IRF6wt. (B) Representative linescan plots (blue: DAPI/nucleus channel; green: FITC/IRF6 channel) of individual cells (left) describing the following three types of IRF6 localization: Type 1: c/n ≥ 1.2; Type 2: c/n = 0.8–1.2; Type 3: c/n ≤ 0.8 (middle). C: cytoplasmic intensity; n: nuclear intensity. Matching IRF6 localizations for each of the three categories are shown as pictures of anti-myc immunostained IRF6-transfected HaCaT cells (right). Scale bar: 10 μm. (C) Dot plots summarizing the analysis of 75 individual IRF6wt- and IRF6_VWS-transfected HaCaT cells by linescan plots showing the c/n ratio. Note that in IRF6_VWS-transfected cells there is a prominent fraction of cells with a ratio c/n ≤ 0.8 (Type 3). ^∗p ≤ 0.001. (D) Relative distribution of exogenous IRF6 localization within transfected HaCaT cells. Note that while most (≈ 80%) of the IRF6wt is localized in the cytoplasm (Type 1: c/n ≥ 1.2), most (≈ 50%) of the IRF6_VWS is found in the nucleus (Type 3: c/n ≤ 0.8).