Minor collagen expression after gain or loss of FRZB in ATDC5 cells. (A) Real-Time PCR analysis for collagen type 3α1 (Col3a1) and collagen type 5α1 (Col5a1) expression in the ATDC5 micro-masses overexpressing frizzled-related protein (FRZB) compared to controls at Day 7. Data are shown as the mean fold difference compared to the control condition normalised to hypoxanthine guanine phosphoribosyl transferase (Hprt) (2-ΔΔCt) ± SEM (n = six samples/condition; Mann-Whitney test: for Col3a1 P = 0.24 and for Col5a1 P = 0.06). (B) RT-PCR analysis for Col3a1 and Col5a1 expression in the ATDC5 micro-masses where Frzb was knocked down using the pGIPZ-shRNAmir directed against Frzb compared to controls at Day 7. Data are shown as the mean fold difference compared to the control condition normalised to Hprt (2-ΔΔCt) ± SEM (n = six samples for pGIPZ-Ctrl and three samples for pGIPZ-FRZB; Mann-Whitney test: for Col3a1 P = 0.047 and for Col5a1 P = 0.54). (C) Proliferation assay of ribcage articular chondrocytes isolated from Frzb-/- compared to wild-type (WT) mice. Data are shown as the difference in fluorescence after 24 h and one week. (n = nine conditions/group; Initial cell densities were 500 (black dots), 2,000 (grey dots) and 4,000 (black circles) cells per well; Mann-Whitney test: P = 0.0019, P = 0.0012 and P = 0.0008).