FIG 4.

DAP5 depletion attenuates global translation and paradoxically induces HIF-1α. (A) HeLa cell lines with dox-inducible eIF4GI or DAP5 depletion were treated with or without dox (96 h), stimulated with TPA as indicated, and treated with PMY (5 μM; 15 min) prior to harvesting. Puromycylation was stopped by washing cells with cold PBS and the addition of lysis buffer. (B) The results of mean quantification of the PMY/tubulin ratio from three experiments are shown, normalizing between experiments by setting the zero-time-point, no-dox value to 1. (C) HEK293 cells with dox-inducible DAP5 depletion combined with mock (−), wt DAP5, or DAP5(E862K) reconstitution were dox treated (96 h) prior to PMY. (D) Mean quantification of the PMY/tubulin ratio is shown for three experiments, normalizing by setting the no-dox ratio to 1. (E) HeLa cells with dox-inducible eIF4GI or DAP5 depletion were dox treated and TPA stimulated, and their lysates were subjected to immunoblotting as shown. (F) Mean HIF-1α, COX2, and MKP-2/tubulin ratios were quantified from three experiments, normalizing by setting the mock (eIF4GI)-depleted, no-dox ratio to 1. All assays were repeated three times, and a representative series for each panel is shown. Error bars denote the SEM. *, significant P value (P < 0.05).