Fig. 2

Hesperetin enhances CISD2, promotes mitochondrial function, alleviates oxidative stress and UVB-induced damage in HEK001 keratinocytes. A Real-time RT-qPCR of CISD2 mRNA in Ker-CT and HEK001 keratinocytes. CISD2 levels were normalized to HPRT1. B Western blot analysis of CISD2 protein in the vehicle (Veh)- and hesperetin (Hes)-treated HEK001 keratinocytes. C The mitochondrial oxygen consumption rates (OCR) of HEK001 keratinocytes after different treatments (n = 10–12). OA, Oligomycin A; Rot/AA, rotenone/antimycin A. D Western blot analysis of CISD2 protein in the shLuc and CISD2 knockdown (KD) HEK001 keratinocytes. E The mitochondrial OCR of HEK001 keratinocytes after different treatments (n = 8). F Intracellular H₂O₂ levels. To assess H₂O₂-induced oxidative stress, HEK001 keratinocytes were treated with 5 μM H₂O₂ for 5 min before monitoring the intracellular H₂O₂ levels. G Mitochondrial membrane potential and quantification of the red/green ratios by JC-1 staining of HEK001 keratinocytes. For hesperetin treatment, 10 μM hesperetin was used to treat the shLuc and CISD2 KD HEK001 keratinocytes for 48 h and mitochondrial membrane potential was monitored before and after 98 μM H₂O₂ treatment. H Protocol for the treatment with hesperetin and its effect on UVB-induced MMP-1 activation in HEK001 keratinocytes. I Western blot analysis of MMP-1 protein in the Veh- and Hes-treated HEK001 keratinocytes after UVB exposure. J ELISA analysis of pro-MMP-1 protein in the conditioned medium from various groups of HEK001 keratinocytes. Vehicle, 0.1% DMSO. All the experiments were performed and repeated three independent times as biological replicates using HEK001 keratinocytes. Data are presented as mean ± SD. In (C and J), the statistical analysis was performed by one-way ANOVA with Bonferroni multiple comparison test. In (E and F), the statistical analysis was performed by two-way ANOVA with Bonferroni multiple comparison test. In (A), (B) and (G), the statistical analysis was performed using Student’s t test; not significant (n.s.)