Enhancement of osteopontin expression in HepG2 cells by epidermal growth factor via phosphatidylinositol 3-kinase signaling pathway

Figure 1 Dose-dependent effect of EGF on osteopontin expres-sion in HepG2 cells. 1×10⁵ cells were plated in 6-well plates. Next morning, cell cultures (near 80% confluent) were incu-bated in serum-free medium for 24 hours. The cells were stimu-lated dose-dependently by epidermal growth factor. Positive control denotes cells in normal growth medium containing 10% fetal calf serum. A. Osteopontin mRNA level was analyzed by RNase protection assay on total RNA using a 486 base-pair probe and controlling the loading with a probe for β-actin. B. Quantitation of osteopontin mRNA levels is shown in (A). RDU ratio reflects relative density units of osteopontin mRNA di-vided by β-actin mRNA. C. The results from RNase protection assay were confirmed by Western blot of osteopontin protein. Tubulin was served as loading control.

Figure 2 Time course showing effect of EGF on osteopontin expression in HepG2 cells. 1×10⁵ cells were plated in 6-well plates. Next morning, cell cultures (near 80% confluent) were incubated in serum-free medium for 24 hours. The cells were stimulated time-dependently by 10 ng/ml EGF. Positive con-trol denotes cells in normal growth medium containing 10% fetal calf serum. A. Osteopontin gene expression was analyzed by RNase protection assay on total RNA using a 486 base pair probe and standardized by comparison to β-actin. B. Quantitation of osteopontin mRNA levels is shown in (A). RDU ratio reflects relative density units of osteopontin mRNA di-vided by β-actin mRNA. C. Western blot of osteopontin pro-tein by cell lysates confirmed the results from RNase protec-tion assay. Tubulin was served as loading control.

Figure 3 Phosphatidylinositol 3-kinase pathway dependent of osteopontin expression in HepG2 cells. The PI3-kinase in-hibitor wortmannin was used to assess EGF-dependent sig-nal transduction leading to osteopontin gene expression. 1×10⁵ cells were plated in 6-well plates. Next morning, cell cultures (near 80% confluent) were incubated in serum-free medium for 24 hours. 100 nM wortmannin was added to the cells 30 minutes before incubation in the presence or absence of 10 ng/ml EGF. A. Osteopontin mRNA level was analyzed by RNase protection assay on total RNA using a 486 base-pair probe and controlling the loading with a probe for β-actin. B. Quantitation of osteopontin mRNA levels is shown in (A). RDU ratio reflects relative density units of osteopontin mRNA divided by β-actin mRNA.