MMP/TIMP knock-downs in hMSCs: effect on mRNA and protein levels. (A) hMSCs were transfected with siRNAs targeting the gene expression of MMP-2 (i), MT1-MMP (ii), TIMP-1 (iii), or TIMP-2 (iv). Control cells were transfected with non–target-directed siRNA (set as 100%). Transcription of specific mRNAs was quantified by qRT-PCR 24 and 72 hours after siRNA transfection. Data represent the mean ± SD of a triplicate measurement representative for 5 transfection experiments. ***P < .001, **P < .01. (B) hMSCs transfected with non–target-directed control siRNA (NC) or with siRNA against MMP-2 (KD) were cultivated under serum-free conditions and analyzed for secreted proMMP-2 after different time intervals by zymography. For densitometric quantification, enzyme release from control cells transfected with non–target-directed siRNA was set as 100% at each time point. (C) Western blotting analysis of protein extracts obtained from hMSCs 72 hours after transfection with control siRNA (NC) or with siRNA against MT1-MMP (KD). Cellular actin was detected on the same blot to control for application of equal amounts of protein in each lane. TIMP-1 and TIMP-2 secretion from hMSCs carrying the respective knock-downs (KD) or control siRNA (NC) was examined by Western blotting of 72-hour culture supernatants. Protein data are representative of 3 independent experiments with similar results.