Expression analyses in differentiated aggregate cultures of human mesenchymal stem cells (hMSCs). Aggregate cultures were prepared and transduced with rAAV-lacZ or rAAV-FLAG-hSOX9, as described in Figure 1, or left untreated, and processed on day 21 for (a) immunodetection of type II, type I, and type X collagen (all at magnification ×4), and (b) gene-expression analysis by real-time RT-PCR amplification after total cellular RNA extraction and cDNA synthesis, as described in Materials and methods. The genes analyzed included the transcription factor SOX9, and types II, I, and X collagen (COL2A1, COL1A1, COL10A1), alkaline phosphatase (ALP), matrix metalloproteinase 13 (MMP13), osteopontin (OP), the transcription factor RUNX2, β-catenin, parathyroid hormone-related protein (PTHrP), lipoprotein lipase (LPL), and the peroxisome proliferator-activated receptor gamma 2 (PPARG2), with GAPDH serving as a housekeeping gene and internal control (primers are listed in Materials and methods). Ct values were obtained for each target and GAPDH as a control for normalization, and fold inductions (relative to untreated aggregates) were measured by using the 2-ΔΔCt method. Statistically significant compared with (a) condition without vector treatment or (b) rAAV-lacZ.