Fig 1.
BBP inhibits mRNA translation in vitro.
Rabbit reticulocyte lysate system was used to measure the translation activity in vitro using luciferase mRNA as the reporter. BBP was added to the final concentrations as indicated. Equal volume of methanol only was used as the control. Treatment with each concentration was repeated three times and the average values were shown. The structure of BBP was inserted in the chart.
Fig 2.
The effects of BBP and MEHP on mRNA translation in HEK-293T cells.
HEK-293T cells were grown to 80% confluence and transfected with luciferase reporter. Eight hours later, cells were treated with BBP or MEHP for 24 hrs at the concentrations indicated. Equal volume of vehicle (methanol) was used as the control. Cells were harvested by washing once with cold PBS, and the cell lysate was prepared with PBL buffer (Promega) for the luciferase activity measurement using dual luciferase assay kit (Promega). (A) Schematic diagram of the reporter construct used in this experiment and throughout the whole study. (B) The effect of BBP on cap-dependent and IRES-driven translation; luciferase activity is the readout of translational activity. (C) The effect of MEHP on cap-dependent and IRES-driven translation. The structure of MEHP was inserted in the chart. The experiments were repeated three times, and the means were shown with standard deviations.
Fig 3.
The effects of BBP and MEHP on mRNA translation in HT29 cells.
HT29 cells were grown to 80% confluence and transfected with luciferase reporter. Eight hours later, cells were treated with BBP or MEHP for 24 hrs at the concentrations indicated. Equal volume of vehicle (methanol) was used as the control. Cells were harvested by washing once with cold PBS, and the cell lysate was prepared with PBL buffer (Promega) for the luciferase activity measurement using dual luciferase assay kit (Promega). (A) The effect of BBP on cap-dependent and IRES-driven translation; luciferase activity is the readout of translational activity. (B) The effect of MEHP on cap-dependent and IRES-driven translation. The experiments were repeated three times, and the means were shown with standard deviations.
Fig 4.
BBP modulates the expression and activity of eIFs in HEK-293T cells.
HEK-293T cells were cultured to 80% confluency and treated with a series of concentrations of BBP for 24 hrs. The cells were harvested and washed once with cold PBS for the preparation of total cell lysate as described in the “materials and methods”. (A) Equal amounts of cell lysate (50μg) were analyzed by Western blot with antibodies against eIFs as indicated. (B) A diagram to show the purification of eIF4F complex by m7GTP-resin affinity chromatography. (C) Analysis of eIF4G and eIF4E in eIF4F complex by Western blot.
Fig 5.
Measurement of binding of BBP to eIF4E by SPR.
Purified eIF4E was immobilized to the Fc2 channel of a CM5 sensor chip as the ligand. The immobilization was performed through amine-coupling reaction. Fc1 channel was treated similarly but without protein as the control. Different concentrations of BBP were applied through Fc1 and Fc2 channels as the analyte to measure the binding. (A) Adjusted sensorgrams (Fc2-Fc1) showing the binding of eIF4E to various concentrations of BBP. (B) Calculation of binding affinity using steady-state fitting model.
Fig 6.
The effects of BBP on cell proliferation.
Both HEK-293T and HT-29 cells were cultured to 80% confluency and treated with BBP at a series of concentrations as indicated. After the treatment for 24 hrs, the cell viability was measured as described in the “Materials and methods”. Each concentration point was repeated three times, and the means were shown with standard deviations.
Fig 7.
Working model of regulation of mRNA translation by phthalates.
The dashed arrows indicate the pathway not studied in the paper.