Fig. 5. Nuclear CLIC4 induces the expression of keratinocyte differentiation markers, and intracellular chloride influences keratinocyte differentiation. (A) Mouse keratinocytes in 0.05 mM Ca²⁺ medium were pre-treated with null, wild-type CLIC4 (Cyt) or nuclear-targeted CLIC4 (Nuc) adenovirus for 12 hours, and cell lysates were analyzed by immunoblots using K1, K10, CLIC4 and actin antibodies. Gray arrow indicates the endogenous CLIC4 (Endo-CLIC4) and the black arrows indicate the two exogenous CLIC4 bands. (B) Mouse keratinocytes were cultured in 0.05 mM Ca²⁺ medium and double-immunostained with CLIC4 and K1 antibodies. Examples of spontaneously differentiating keratinocytes are indicated by white arrows. All immunostained cells were analyzed by confocal microscopy as described in the Materials and Methods. Insets show DAPI staining. (C) Mouse keratinocytes in 0.05 mM Ca²⁺ medium were infected with adenoviruses encoding -gal, wild-type CLIC4 (Cyt-CLIC4), CLIC4 (Nuc-CLIC4) or nuclear-targeted GFP (Nuc-GFP) for 16 hours, treated with MQAE fluorescent dyes and assayed by confocal microscopy. (Insets) The chloride ion content of the -galactosidase, Cyt-CLIC4 or Nuc-CLIC4 Ad infected cells treated with NPPB was visualized by confocal microscopy. (D) The fluorescence intensity was also measured in similarly treated cells. Fluorescence on the y axis is shown in arbitrary units and decreasing values (quenching) indicates greater Cl^– content. (E) Mouse keratinocytes were treated with increasing amount of NPPB (gradient triangle, 200 µM for 1x and 2 mM for 10x) for 2 hours prior to calcium (0.5 mM) induction. After 24 hours, cell lysates were analyzed by immunoblots using K10, CLIC4 and actin antibodies.