Figure 3.

Filaggrin-2 knockdown leads to impaired stratum corneum properties. (a and b) A solution of Lucifer yellow was applied to the shc- and shFLG2-RHEs. After 6 h of incubation, location of the dye was investigated using a fluorescence microscope (a) and dye concentration in the culture medium was quantified (b). As a control, a normal RHE at day 4 was used (RHE-D4). (c) The pH was measured at the surface of the RHEs. Each indicated value corresponds to the mean pH of a different RHE (n=5 shc-RHEs and n=6 shFLG2-RHEs). The RHEs were produced with keratinocytes from two different donors. (d) Pyrrolidone carboxylic acid (PCA) and urocanic acid (UCA) amounts were quantified in lysates of shc-RHEs (n=8) and shFLG2-RHEs (n=9). RHEs were produced with keratinocytes from the same two different donors. (e) Active caspase-3 (red staining; arrows) was detected without and with UVB irradiation, as indicated. Please note the nonspecific labeling of the polycarbonate membrane (under the thin line). (f) The number of active caspase-3-positive cells was quantified and is indicated per RHE length unit. Differences in pH, in amino-acid amounts and in number of cells were analyzed using Student's t-test. Only statistically significant differences are indicated