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. 2012 Jul 26;8(7):e1002833. doi: 10.1371/journal.ppat.1002833

Figure 3. Differentiation-specific nuclear C/EBPβ expression in human epidermis and C/EBPβ-mediated induction of CCL20 expression in NHK.

Figure 3

(A) Sections of normal human skin were stained using a polyclonal antibody against C/EBPβ (brown) and hematoxylin. A higher magnification of the complete epidermis is presented. Bars correspond to 100 µm and to 50 µm in the higher magnification. (B) NHK were co-transfected with the CCL20 promoter reporter construct and increasing amounts of C/EBPβ expression vector as indicated. Total amount of DNA was adjusted with the pcDNA3.1+ control vector. After 24 h the luciferase activity was measured and normalized to protein concentrations of the respective luciferase extracts. The normalized luciferase activity of the control transfection was set at 1. Shown are the values averaged from two transfections performed in triplicates ± SD. (C, D) RTS3b cells were transfected with C/EBPβ expression vector. After 24 h mRNA was isolated from the cells and quantified by real-time PCR for CCL20 in relation to GAPDH (C). Supernatants collected from these cell cultures were assessed for CCL20 protein expression by ELISA (D). Shown are the mean values ± SD from at least three independent experiments. Asterisks represent statistical significances, p<0.001. (E) Two promoter-proximal C/EBP binding sites are crucial for CCL20 activation. NHK were transfected with luciferase reporter constructs either under the control of the wild-type CCL20 promoter (black bars) or the CCL20 promoter mutated in one (white bars) or two (grey bars) proximal C/EBP binding sites and co-transfected with C/EBPβ (400 ng) expression vector. Total amount of DNA was adjusted with the pcDNA3.1+ control vector. After 24 h the luciferase activity was measured and normalized to protein concentration of the respective luciferase extracts. The normalized luciferase activity of the control transfection was set at 1. Shown are the values averaged from three transfections performed in triplicates ± SD. Asterisks represent statistical significances, p<0.0001.