MRSA co-culture induces TLRs expression by hMSCs. Human MSCs total RNA was subjected to RT-PCR and qRT-PCR using SYBR green assay with TLRs specific primes. Representative RT-PCR of each TLR was shown. Human MSCs with no bacterial infection cDNA was used as control. U937 cell line cDNA was used as positive control for PCR amplification (A). GAPDH was used as a reference. The relative mRNA expression was determined by the delta-delta comparative threshold (ddCT) method. (B). TLR1 and TLR2 protein expression in MSCs are shown (C). Cytokines stimulation in hMSCs by MRSA. RT-PCR was done with human specific primers of IL-8, IL-6, NR4A2, VDR, osteopontin (OPN), and GAPDH as a control (D). RT-PCR data in hMSCs were further confirmed with qRT-PCR analysis using SYBR green assay, where GAPDH as an endogenous control (E). Data are from n = 3 independent experiments done in triplicate. *P < 0.05 compared to control. MSCs were co-culture with and without S.aureus and cultured in osteogenic medium supplemented with dexamethasone (0.1μM). Alizarin Red staining was done at day 15. Alkaline phosphatase activity was measured at day 9 from cell lysate as released p-nitrophenol. At day 12, cells showed change in morphology and bone nodule formation in cultures of differentiating osteoblasts. Representative picture was shown. Original magnifications are 10× (F). Cells were incubated with DRAQ5 for 5 min followed by washing with PBS and scanned in Li-Cor Odyssey Infrared Scanner. Equal area scanned intensities were plotted against samples of different day of treatment (G). n = 3, * P < 0.002 compared to control. Western blot analysis of Cyclin D1 using Day12 and Day15 MRSA co-culture hMSCs protein extracts compared to Day 0, and Day 12 control extracts (H).
Maiti and Jiranek BMC Cell Biology 2014 15:11 doi:10.1186/1471-2121-15-11