Chondrogenesis after gain or loss of FRZB in ATDC5 cells. (A) Real-Time PCR analysis showed increased expression of collagen type 2a1 (Col2a1) and aggrecan in the micro-masses overexpressing frizzled-related protein (FRZB) compared to micro-masses expressing control pcDNA3.1+ vector. Data are shown as the mean of the fold difference compared to the control condition at Day 7 normalised to hypoxanthine guanine phosphoribosyl transferase (Hprt) (2-ΔΔCt) ± SEM (n = six samples/condition; Mann-Whitney test: for aggrecan P = 0.002, P = 0.015 and P = 0.002 and for Col2a1 P = 0.03, P = 0.3 and P = 0.002). (B) Picrosirius Red and Safranin O staining at Day 7 showed increased spreading of collagen fibers and sulphated glycosaminoglycans (GAGs) from the center in micro-masses overexpressing FRZB compared to controls. (C) Protein quantification (optical density (OD) measured at 570 nm) of the micro-masses was comparable between the two groups (n = three samples/group; Mann-Whitney test: P > 0.05). (D) Staining intensity was comparable between FRZB overexpressing micro-masses and controls for Picrosirius Red and significantly decreased for FRZB overexpressing micro-masses for Safranin O staining. Data are shown as the mean OD normalised to the mean protein content (n = three samples/group; Mann-Whitney test: P > 0.05 for Picrosirius Red and P = 0.02 for Safranin O). (E) Real-Time PCR analysis showed decreased expression of Col2a1 and aggrecan in the micro-masses where Frzb was knocked down using the pGIPZ-shRNAmir directed against Frzb compared to controls. Data are shown as the mean of the fold difference compared to the control condition at Day 7 normalised to Hprt (2-ΔΔCt) ± SEM (n = two to three samples for pGIPZ-Ctrl and five to six samples for pGIPZ-FRZB; Mann-Whitney test: for aggrecan P = 0.02 and for Col2a1 P = 0.02). At Day 14 and Day 21 for pGIPZ-Ctrl n = 2 precluding statistical analysis.