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Fig. 6. Kazrin induces terminal differentiation. (A-D) Still frames from a time-lapse recording (see Movie 1 in supplementary material) of keratinocytes transiently expressing GFP-kazrinA (green). The times in each frame are minutes of recording. Black and white arrows follow the paths of two transfected cells. (E,F) Vector diagrams tracking migration of (E) ten untransfected and (F) ten GFP-kazrinA-expressing cells in Movie 1 in supplementary material. (G) Clonogenicity assay in which 10³ keratinocytes transduced with either GFP or HA-kazrinA were plated onto mitomycin-C-treated J2 feeders. Two weeks post-plating, cells were fixed and stained with Rhodamine B (left panels) or examined by phase-contrast microscopy (right panels). (H,I) Single confocal slice images of keratinocytes expressing GFP (H; green) or HA-kazrinA (I; green), labelled with antibodies specific for involucrin (red) 48 hours post transfection. Bars, 200 µm (A-D,G), 100 µm (H,I). (J-L) Flow cytometric analysis of keratinocytes transduced with GFP or HA-kazrinA (KA), plated in the absence of feeders, and incubated for 4 days in low Ca²⁺ (J, upper panels) or standard medium (J, lower panels; K,L). Cells were gated on GFP- or HA-positive populations and then analyzed for forward and side scatter (J), or expression (dark lines) of involucrin (K) or transglutaminase 1 (L). Grey lines show control staining with secondary antibody alone (K,L). Percentages of cells in gated regions are indicated.

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