Figure 4
1,25(OH) 2 D3 pretreatment increases global histone H3 methylation (H3K9me3) to reduce MRSA-mediated cytokines synthesis. Human MSCs were pretreated 24 hrs with 1,25(OH)2D3 (100 nM) followed by co-culture with methicillin-resistant Staphylococcus aureus (MRSA) for another 24 hrs and harvested for total cellular proteins extraction. Equal amount of proteins were used for western blot analyses with indicated antibodies (A). Experiments were done three times and representative blots were shown. HEK293 cells (B), and human MSCs (C) were treated with or without 5Aza deoxycytidine (AZA) for 2 hrs followed by with or without 24hrs preincubated with 1,25(OH)2D3 (100 nM) followed by methicillin-resistant Staphylococcus aureus (MRSA)-stimulation for 24 hrs. Total RNA from hMSCs was isolated and processed for cDNA synthesis. Real-time quantitative PCR was performed with human specific primers of IL-8, using SYBR green assay. Endogenous control human GAPDH was used as a reference. The relative mRNA expression was determined by the delta-delta comparative threshold (ddCT) method. Data are from n = 3 independent experiments done in triplicate. * P < 0.02 for VitD + MRSA vs.VitD + AZA + MRSA stimulation, ** P < 0.0002 for MRSA vs. VitD + MRSA stimulation.