Constitutive expression of mRNA and protein of MMPs and TIMPs in hMSCs. (A) qRT-PCR analysis of MMP-2, MMP-9, MT1-MMP, TIMP-1, and TIMP-2 gene transcription in hMSCs cultivated in MSCG medium. The results are mean values ± SDs of mRNA expression relative to GAPDH (set as 1) from a triplicate measurement representative for 3 independent experiments with 3 different hMSC lots. (B) Western blot detection of MMP-2, MMP-9, MT1-MMP, TIMP-1, and TIMP-2 in cell lysates (LY) and conditioned medium (CM) of hMSCs cultivated for 72 hours under serum-free conditions. Aliquots (30 μL) standardized by protein content were separated by SDS-PAGE under reducing conditions, blotted, and probed with the specific antibodies. (C) Zymographic analysis of gelatinase secretion from hMSCs. Aliquots of culture supernatants (10 μL) taken at different time points during a 72-hour cultivation period of hMSCs in serum-free medium were analyzed. HT1080 conditioned medium containing proMMP-9, proMMP-2, and active forms of MMP-2 was used as a marker (M).³⁷