Figure 5.

Caspase-14 induces DNA degradation via the ICAD–CAD system. (a) Western blot analysis of ICAD degradation products. Human caspase-14 was purified from corneocyte extract of healthy individuals using sequential chromatographic procedures.¹⁷ Purified active caspase-14 was incubated with ICAD, and ICAD degradation products were subjected to SDS-PAGE (15 μg each). Western blot analysis was carried out using anti-ICAD antibody (FL331). Western blot analyses of cornified cell extract (CC), ICAD+purified caspase-14 (30 min and 3 h incubation) and ICAD+caspase-3 (30 min incubation) are shown. (b) ICAD degradation by constitutively active caspase-14 (revC14). The effect of zVAD-fmk is also shown. (c) Effect of kosmotropic ion (sodium citrate) on ICAD degradation. ICAD degradation by purified caspase-14 was tested with or without kosmotropic ion. (d) Effect of kosmotropic ion on caspase-3 activity. ICAD-degrading activities of caspase-3 were examined with or without kosmotropic ion. (e) Accumulation of CAD after revC14 transfection. Keratinocytes were transfected with pInv-HA-revC14 and 2 days after confluency, examined with anti-CAD Ab and TUNEL staining. (f) Colocalization of CAD and revC14. The presence of revC14 was verified with anti-HA staining. Scale bars, 50 μm. (g) Association of active caspase-14 and ICAD in normal epidermis. PLA was carried out using antibody combinations with h14D146 and anti-ICAD Ab. Normal rabbit IgG was used as negative control. A merged figure with nuclear staining (DAPI) is also shown. The broken line indicates the epidermal–dermal junction. The dotted line shows the border of the cornified layer. Blue staining (DAPI) indicates nuclei. Scale bars, 50 μm