Fig. 2.

Cutibacterium acnes upregulates the mRNA and protein expression of tumour necrosis factor alpha-induced protein 3 (TNFAIP3). (A–F) Human immortalized keratinocyte cell line (HPV-KER) cells and (G, H) organotypic skin (OS) were treated with C. acnes, and changes in TNFAIP3 mRNA and protein levels were analysed. (A, B) mRNA expression was analysed by real-time RT-PCR; data were normalized to 18S rRNA and compared with the time-match untreated control values. Error bars: standard error of the mean (SEM). (C, D) TNFAIP3 protein expression was analysed by western blot and quantified using Image Pro Plus (Media Cybernetics, Inc., Rockville, MD, USA); all data were normalized to actin and compared with time-matched untreated control values. Statistical analyses: paired, 2-sample t-test with Holm–Bonferroni correction: *p < 0.05. (E, F) Cells were plated and pretreated with selective inhibitors for Jun kinase (JNK), nuclear factor kappa B (NF-κB), p38 and ERK1/2 or, as a control, Dimethyl sulphoxide (DMSO) for 1 h. For mRNA expression studies, C. acnes challenge was performed for 12 h and analysed by real-time RT-PCR. All data were normalized to the 18S rRNA and compared with DMSO-treated control samples. For protein expression analyses, cells were treated with C. acnes for 24 h, subjected to western blot analysis and quantified with Image Pro Plus; all data were normalized to actin and compared with time-matched untreated control values. Statistical analyses: paired, 2-sample t-test with Holm–Bonferroni correction, in which basal TNFAIP3 expression, *p < 0.05, or C. acnes-induced TNFAIP3 expression, #p < 0.05, were compared. (G, H) OS cultures were treated with 3×10⁷ bacterium/cm² for 24 h. The epidermis was separated by dispase digestion and the mRNA expression was analysed only in epidermal cells by real-time RT-PCR. Protein expression was visualized using immunofluorescence staining for TNFAIP3 (green) and DAPI (blue) in the full-thickness skin biopsy.