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. 2015 Jun 8;6(27):24075–24091. doi: 10.18632/oncotarget.4373

Figure 4. Doxorubicin and Madcam1 had opposite effects on eIF4E.

Figure 4

A. Doxo reduced 4EBP1 phosphorylation. Phosphorylation of translation-associated proteins was detected in Bel-7402 cells treated with DMSO or Doxo (final concentration 2.0 μg/ml) for 4 h before harvest for WB using the indicated antibodies. B. p-4EBP1 and 4EBP1 were detected by WB in Bel-7402 cells treated with DMSO or Doxo (final concentration 2.0 μg/ml) for 4 h before harvest. C. Madcam1 overexpression induced 4EBP1 phosphorylation, as detected by WB, in Bel-7402 cells transfected with empty or Madcam1-Myc expressing plasmids. D. Madcam1 knockdown reduced 4EBP1 phosphorylation, as detected by WB, in Bel-7402 cells infected with GFP-sh, Madcam1-sh1 or Madcam1–sh2. E. Doxo induced 4EBP1 binding to eIF4E. Bel-7402 cells were treated with DMSO or increasing concentrations of Doxo (final concentration from 0.5-2.5 μg/ml) for 5 h before harvest in IP lysis buffer. Endogenous eIF4E was then immunoprecipitated with an anti-eIF4E antibody and 4EBP1 co-immunoprecipitation was detected by WB. F. Madcam1 reduced 4EBP1 binding to eIF4E. Endogenous eIF4E was immunoprecipitated with an anti-eIF4E antibody and 4EBP1 co-immunoprecipitation was detected by WB for control (transfected with Empty vector) and Madcam1-Myc transfected Bel-7402 cells, using increasing amounts of ectopically expressed Madcam1-Myc. G. Identification of the subcellular localization of Madcam1. Co-localization of Madcam1 and possible organelles was analyzed in Bel-7402 cells by immunofluorescence assays using a combination of anti-Madcam1 (red) and anti-LC3B (autophagosome marker, green), anti-COX IV (mitochondria marker, green), anti-Calnexin (endoplasmic reticulum (ER) marker, green) or anti-NUP98 (nuclear envelop marker, green) antibodies. The area indicated by the square was enlarged on the right side. Scale bar, 10 μM. H. Madcam1 reversed Doxo-induced dephosphorylation of 4EBP1. Bel-7402 cells were treated with or without Doxo (final concentration 2.0 μg/ml) for 4 h in the presence or absence of ectopically expressed Madcam1-Myc before harvest for WB using anti-p-4EBP1 and 4EBP1 antibodies. I. Madcam1 prevented Doxo-induced protein down-regulation. Bel-7402 cells were treated as described in Figure 4H and were harvested for WB using the indicated antibodies. J. Madcam1 inhibited Doxo-induced dissociation of eIF4E from the mRNA. The RNA was extracted from Bel-7402 cells that were treated as in Figure 4h for RNA-IP assays using anti-eIF4E antibodies. The data from the “Mock” group were arbitrarily set to 100 %. The data are shown as the means + SD from three independent experiments (including WB). *, p < 0.05 using the Student's t test.