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Fig. 3. CLIC4 upregulation is required for Ca²⁺-induced differentiation in keratinocytes. K1, K10, Filaggrin, CLIC1, CLIC4, PKC and actin protein levels in mouse keratinocytes were determined by immunoblot analysis. (A) Mouse keratinocytes were co-infected with 5 MOI of Tet-Off Ad (to induce expression of the conditional AS) and increasing MOI (5 and 10, shaded triangle) of AS-CLIC4 Ad or Null Ad (N) for 17 hours before treatment with 0.5 mM Ca²⁺ for 24 hours. (B) Duplicate sets of mouse keratinocytes were treated with BrdU, and anti-BrdU-FITC stained cells of each sample were analyzed with flow cytometry. Cell populations: null Ad-infected without BrdU (upper left, N/(–)/Lo Ca²⁺), with BrdU (upper right, N/(+)/Lo Ca²⁺) both in 0.05 mM Ca²⁺; with 0.5 mM Ca²⁺ and BrdU (lower left, N/(+)/Hi Ca²⁺); co-infected with AS-CLIC4 Ad and Tet-Off Ad followed by Ca²⁺ and BrdU treatment (lower right, AS/(+)/Hi Ca²⁺). (C) The key indicates the gated population in each cell cycle phase. The percentage of cells in each cell cycle phase from the total cell population in N/(+)/Lo Cal²⁺, N/(+)/Hi Ca²⁺ and AS/(+)/Hi Ca²⁺ at 24 hours and 48 hours after Ca²⁺ induction is represented in a bar graph format. (D) Human 293 cells were transfected with constructs expressing nonspecific (NS) or CLIC4 (sR-CLIC4) shRNA for 48 hours, and specificity of CLIC4 knockdown was analyzed by immunoblots of cell lysates. (E) Mouse keratinocytes were transfected with constructs expressing shRNA for nonspecific (NS), CLIC1 (sR-1), CLIC4 (sR-4) or CLIC5 (sR-5) for 24 hours, and then treated with 0.05 mM (Lo) or 0.5 mM (Hi) Ca²⁺. Actin was used as a loading control. Similar data were obtained from at least two independent experiments. The numbers over the CLIC4 bands in A and B represent fold changes determined by densitometry.

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