Alpha-actinin was used to identify cardiomyocytes while the β-subunit of prolyl 4-hydroxylase was used as a marker for cardiac fibroblasts. The left panels show the results of western blots with the representative bands and their average intensity values normalized to respective controls. The right panels show the representative confocal images for each marker.
A. 50μg/ml DEHP-treated samples had evenly spaced α-actinin staining, while control sample cells tended to bundle into clusters. The latter is consistent with the changes occurring in long-term cultures of neonatal cardiomyocytes (note: samples were analyzed on day 8 after cell plating). The amount of total protein in Western blots (n=6) was slightly increased in DEHP-treated samples (p=0.015).
B. Western blot assays and the distribution of fibroblast marker prolyl 4-hydroxylase, in control and 50μg/ml DEHP-treated samples. No difference between control and DEHP-treated samples was observed (n=8).