Figure 2
Enhanced chondrogenesis in pellets of AF cells by knockdown of Pax1. (a) AF cells isolated from 4-week-old Wistar rats were seeded at a density of 8 × 104 cells/well in a 12-well plate. Morphology of AF cells cultured in DMEM containing 10% FBS at day 1 is shown. (b) Total RNA was extracted from AF cells maintained in a culture dish for 96 h and then processed for RT-PCR analysis of expression of ECM molecules (Acan, Col1a2, Col2a1, Ocn) and transcription factors (Pax1, Pax9, Nkx3.2, Sox5, Sox6, Sox9). Full-length gels are presented in Supplementary Fig. 7. (c) AF cells were seeded at a density of 4 × 104 cells/well in a 24-well plate. At 24 h after inoculation, the cells were transfected with non-targeting siRNA (control) or Pax1 siRNAs (siPax1-a or siPax1-b) by lipofection. Total RNA was extracted from the cells at 48 h after lipofection, and the expression levels of Pax1 or Acan were examined by qRT-PCR. (d) Experimental schedule of chondro-induction in pelleted cultures of AF cells. AF cells were seeded at a density of 1 × 105 cells/well in 6-well plates and then infected with shRNA lentiviruses on day 1. After trypsinization on day 3, cell pellets were prepared from 1.5 × 105 cells/pellet and cultured in chondrogenic differentiation medium for another 21 days. (e–g) Sections of cell pellets infected with shRNA lentiviruses were stained with alcian blue. Increased accumulation of alcian blue-stained extracellular matrix was observed in the pellets of AF cells infected with shPax1-a or shPax1-b lentivirus (f,g) compared to that in cells infected with control lentivirus (e). (h) Total RNA was extracted from pellets on day 24. Relative expression levels of Pax1, Sox9, and Acan were examined by qRT-PCR analysis. qRT-PCR data (c,h) represent the average of three independent experiments. The relative expression of each gene is normalized to that in the control and reported as mean ± s.d. ***P < 0.001 versus control. Scale bars in (a) and (e–g), 100 μm.
