Fig. 4. CLIC4 translocates to the nucleus in differentiating and senescing keratinocytes in vitro and is primarily nuclear in epidermis in vivo. (A) Mouse primary keratinocytes and human foreskin keratinocytes were treated with indicated Ca²⁺ for 24 hours and immunostained with CLIC4 antibody. (B) Mouse keratinocytes were maintained in 0.05 mM Ca²⁺ medium and either fixed on the first day of culture (1 Day) or after 7 days of culture (7 Day) to allow for senescence. Fixed cultures were immunostained with CLIC4 antibodies. Duplicate sets of cells were subjected to assay by -gal SA, and the number of senescing cells (7 Day) is presented as a fold increase over the control (1 Day; lower panel). (C) Mouse keratinocytes were incubated in the serum-depleted medium for 16 hours (0 min), rinsed and treated with the serum-containing medium (30 min). (D,E) Mouse keratinocytes were treated with TGF (1 ng/ml; D) or TPA (25 ng/ml; E) for the times indicated and immunostained for CLIC4. Insets in Fig. 4C-E represent DAPI nuclear staining. (F) Formalin-fixed human breast skin and acetone-fixed mouse skin frozen sections were stained with CLIC4 antibodies by immunohistochemical methods as described in the Materials and Methods. The mouse skin section was from a hyperplastic area to enhance the resolution of strata. Red and green arrows indicate keratinocytes in the basal layers (blue nucleus) and the differentiating layers (brown nucleus, CLIC4 stain) respectively. In all immunofluorescent and immunohistochemical staining experiments, secondary antibody alone was used as controls.