Figure 1.

Stability of site-specific gene expression in fibroblasts. (A) Stability of HOX gene expression after serial passage. The expression level of HOX genes plotted for passage 5 (X-axis) and passage 35 (Y-axis) for thigh (left) and foot fibroblasts (right). (B) Stability of site-specific gene expression patterns with heterotypic conditioned media. Unsupervised hierarchical clustering of thigh fibroblasts (P) without coculture (−), cultured with their self-conditioned media (P, P) or with conditioned media from foot fibroblasts (P, D) and vice versa for foot fibroblasts. Expression level of each gene above (red) or below (green) the global median (black) is denoted by the color scale (fourfold to 0.25-fold on linear scale or +2 to −2 on log₂ scale). (C) UPRT-mediated RNA capture. Only UPRT⁺ cells can incorporate 4TU into thio-uracil (sU) in RNA, enabling retrieval of cell-type-specific RNA by thio-directed RNA biotinylation and purification. (D) UPRT-mediated RNA biotinylation. The 18S RNA band is shown by Northern blot probed with streptavidin-horseradish peroxidase (top) or ethidium bromide (bottom). (E) Verification of cell-type-specific RNA capture from 1:1 mixture of UPRT⁺ and UPRT⁻ RNA. In the absence of the biotinylation reaction, little RNA was retrieved from either population. With biotin-mediated RNA pull down, RNA from UPRT⁺ cells was specifically enriched by eightfold to 10-fold compared with that of the mixed population. (F) Little change in the expression of genes diagnostic of proximal–distal limb origin in human foot fibroblasts after coculture with thigh fibroblasts. (G) Synergistic effects of trichostatin A (TSA) and Aza-C (Aza) on HOXA13 mRNA expression.