Growth responses of Pln knockdown clones. A. Growth curves for murine C3H10T1/2 embryonic fibroblasts stably transfected with control ribozymes or those specific to Pln were performed as described in “Materials and Methods”. Cell numbers were determined by hemocytometer counting in triplicate cultures at each time point. The data represents the average cell density for control clone 16 (CR16), and Pln knockdown clones PR12 and PR18, during 14 days of culture. All clones share similar growth patterns. B. BMP-2 growth responses of Pln knockdown clones were determined as described in “Materials and Methods”. The bars represent the mean (+/- standard deviation) cell density of triplicate determinations of BMP-2 (100 ng/ml) treated (white bars) and untreated (black bars) clones determined on day nine of culture. All cells responded to BMP-2 supplementation to a similar extent, and similar effects were observed at earlier culture times.

Indirect immunohistochemical analysis of micromass cultures treated with BMP-2. Micromass cultures were prepared with and without BMP-2 (100 ng/ml) treatment for ten days, and immunostained as described in “Materials and Methods”. Panels A1, B1, C1, D1, E1, and F1 are from high density micromass cultures in the presence of BMP-2, while panels A2, B2, C2, D2, E2, and F2 are culture in the absence. The top six panels (A1-C2) are from cultures stained with antibodies specific to aggrecan, and the bottom six panels (D1-F2) are from cultures stained with antibodies to collagen type II and counter stained with the nuclear dye Syto13. Note that the signal intensity for aggrecan and collagen type II in the control clone (CR16) is enhanced relative to untreated cultures (A1 vs. A2 and D1 vs. D2). Aggrecan and collagen type II signal intensities in the control clone (CR16) treated with BMP-2 also are elevated above BMP-2 treated knockdown clones PR18 and PR12 (A1 vs. B1 and C1, and D1 vs. E1 and F1). All images are optical sections, captured with a laser scanning confocal microscope under identical settings. (magnification = 20X).

Immunohistochemical analysis of chondrogenic markers in C3H10T1/2 cell condensations formed on PlnDI and treated with BMP-2. A. On days 3, 6, and 12 of culture, both BMP-2 treated and untreated PlnDI-stimulated nodules were collected and labeled with PNA, or antibodies specific to collagen type II, collagen type X, or Pln as described in “Materials and Methods”. In day 3 condensations treated with BMP-2 the signal intensities for PNA (green) and collagen type II (red) are enhanced relative to untreated condensations (panels A vs. B, and C vs. D, respectively). The intensity of Pln labeling (red signal) in treated and untreated condensations is similar (panels E vs. F). Relative to day 3 condensations, the signal for PNA binding in day 6 condensations is reduced (day 6, panel A and B). The signal intensity for collagen type II in untreated condensations is elevated (day 3 D vs. day 6 C), but not different from treated day 6 condensations (day 6 panel C vs. D). The signal intensity for Pln is similar in treated (day 6 E vs. day 3 E), but reduced in untreated condensations (day 6 F vs. day 3 F and day 6 E). In day 12 condensations the signal intensities for collagen type II and Pln are reduced relative to condensations from day 3 and 6 cultures. BMP-2 treated condensations maintain a higher signal intensity for collagen type II and Pln relative to untreated condensations (C vs. D; and E vs. F). Day 12 condensations treated with BMP-2 express high levels of collagen type X (red signal, A). Untreated condensations do not express collagen type X (B). B. Representative images of collagen type X expression (red signal) in condensations from control and Pln knockdown clones following 10 days of BMP-2 treatment. In all panels the blue signal represents DAPI labeling of cell nuclei. All images were captured under identical conditions, employing the same magnification as indicated by the bar in panel D (bar in 6A = 50 μm, and in 6B = 100 μm).

Quantification of Pln secretion. Dot blot analysis of Pln secretion by C3H10T1/2 murine embryonic fibroblasts stably transfected with ribozymes specific for Pln. Conditioned medium was blotted onto nitrocellulose and probed with Pln specific antibodies. Following exposure to film, blots were scanned by densitometry, and individual density values were expressed relative to cell number and averaged. A standard curve employing known amounts of Pln was used to quantify the amount of Pln bound. C3H10T1/2, parental cell line; CR14 and CR1 C3H10T1/2 cell clones stably transfected with the control ribozyme; PR12 and PR18 C3H10T1/2 cell clones stably transfected with Pln ribozymes. The secretion of Pln is reduced ∼70 and 60 % in knockdown clones PR12 and PR18, respectively. These data represent the mean +/- S.D. of triplicate determinations in each case (^*P<0.01).

Alcian blue staining of high density micromass cultures. Alcian blue staining of micromass cultures was performed following ten days of culture with and without BMP-2 (100 ng/ml), as described in “Materials and Methods”. Micromass cultures containing cells from the ribozyme control clone, CR16 demonstrate strong Alcian blue labeling (panel A) following BMP-2 treatment. In contrast, Pln knockdown clones (PR12 and PR18) stain weakly for Alcian blue, and demonstrate a different labeling distribution relative to control (panels C and E vs. A). Alcian blue staining in control clones is intense and localized to the entire micromass; however, knockdown clones demonstrate weak or no labeling in the center and the majority of the staining on clone PR12 is at the periphery, surrounding the micromass. Data presented are representative images collected from triplicate cultures

Oil Red O staining of high density micromass cultures. Monolayer cultures were maintained for ten days in the presence of BMP-2 (100 ng/ml) prior to staining with Oil Red O as described in “Materials and Methods”. Panels A and B represent control clones CR14 and CR16, respectively, and are negative for Oil Red O. Panels C and D, represent clones PR18 and PR12, respectively, and are positive for Oil Red O. Major areas of lipid droplet accumulation are denoted by black arrows. Clone PR12 has a greater abundance of lipid filled cells than clone PR18. All images were captured under the same magnification as indicated by the bar in panel D (bar= 50 μm). Data presented are representative images collected from triplicate cultures.