Morphometric Analysis and Quantification

Using ImageJ software, the mean percentages of IFT140-positive regions, both in growth plate chondrocytes and in trabecular osteoblasts, were calculated in 10 random fields (~20 cells in each field) for each immunofluorescence image. The positive signal threshold was set according to the signal intensity of the respective negative control image. The ratio of IFT140-positive signal in cells was calculated by dividing the measured IFT140-positive area by the total area of the cells (prohypertrophic chondrocytes and osteoblasts). To facilitate the illustration of IFT140 expression change at different growth stages and locations in the long bone, thresholds of 10%, 30%, 50%, and 70% were used to classify IFT140 expression levels into several grades: a ratio greater than 70% (++++), between 70% and 50% (+++), between 50% and 30% (++), between 10% and 30% (+), and less than 10% (+/–).