Figure 4.

Silibinin inhibits MCP-1 transcript level as well as DNA binding of transcriptional regulators of MCP-1 in CAFs. CAFs were incubated in basal media in the presence or absence of silibinin (90 µM) for the indicated times. (A) At the end of each time point, cells were collected and total RNA isolated. Real-time PCR was performed to measure silibinin effect on MCP-1 mRNA as detailed in Methods. Data were normalized against glyceraldehyde 3-phosphate dehydrogenase (*P ≤ 0.05). (B–D) Nuclear lysates were collected and analyzed by EMSA and supershift assay to define silibinin (90 µM) effect on MCP-1 transcriptional regulators NF-κB and AP-1 after 24–72h of treatment. Gels cropped for clarity. Densitometry values, shown under the gels in panels (B) and (C), were determined by normalizing band intensities against background and expressed as fold of control values independently for each time point of the study.