TGF-β1 induces cytokines MIP-1α and MCP-1 that participate in cell migration. (A) Cells were incubated with or without 10 μg/mL CHX, 50 ng/mL ActD, and 30 μM H89 for 30 minutes and then with or without TGF-β1 for 1 hour or 24 hours. Cells pretreated with TGF-β1 for 1 hour or 24 hours were washed, resuspended in 200 μL DMEM-12, and then loaded in the upper reservoir. Then the lower reservoir was filled with 1 mL DMEM-12 containing 5 ng/mL TGF-β1, and migration of the cells was determined as described in the legend to Figure 1. The results are means ± SE of 3 independent experiments, and each value was compared to the corresponding control (none, Tat-C3 = 0 μg/mL) at the same time scale in the presence of TGF-β1 as a chemoattractant (*P < .05 and **P < .01). (B) Cells were treated with 5 ng/mL TGF-β1 and 1 μg/mL LPS for indicated times. The levels of MIP-1α and GAPDH mRNAs were measured by RT-PCR. (C) Cells were preincubated with or without 10 μg/mL CHX, 50 ng/mL ActD, 30 μM H89, or 30 μM PD98059 for 30 minutes and then with 5 ng/mL TGF-β1 for 1 hour or 24 hours. Levels of MIP-1α and GAPDH mRNA were determined by RT-PCR. (D) MIP-1α and MCP-1 secreted from Raw 264 cells in response to 5 ng/mL TGF-β1 at various time points were measured by ELISA technique. The results are means ± SE of 3 independent experiments. (E) Antibodies (4 μg/mL) against p21WAF (cyclin-dependent kinase inhibitor), MIP-1α (I), and MCP-1 (C) were added to both upper and lower chambers. Anti-p21WAF antibody was used as a control. Cells pretreated with 5 ng/mL TGF-β1 for 1 hour or 24 hours were loaded in the upper chamber, and then cell migration was estimated in the presence of 5 ng/mL TGF-β1 in lower chamber as in Figure 1. The results are means ± SE of 3 independent experiments (**P < .01).