Fig 1: eIF2A and eIF4A involve in VHLα alternative translation.a Immunoblots analysis of ectopic VHLα in 293T cells in response to synonymous mutations introduced at CTG43 (left) and neighboring codons (AGG34 > AGA, ATC37 > ATA, CTT40 > CTA, CGC46 > CGT, ACG49 > ACT and CGC52 > CGG, right). b Quantitative PCR measurements of relative expression of eIF2A in established stable cells (786–0 with empty vector (E.V) or HA-eIF2A, 769-p with shNT, sheIF2A-1 and sheIF2A-2). c Immunoblots analysis of VHLα in response to eIF2A overexpression (left) or knockdowns (right) in 293T. d Time course of VHLα induction in 293T cells by aurin tricarboxylic acid (ATA, 100 αM) treatments for 0, 6, 12, 24 h. e VHL mRNA transcript levels were determined by real-time PCR in ATA-treated 293T cells at 0, 6, 12, and 24 h. f Immunoblots analysis of VHLα and VHL in response to different concentrations of scriflavin treatments (0, 10, 20, 30 M) for 4 h in 293T cells. g VHL mRNA transcript levels were determined by real-time PCR in acriflavin-treated 293T cells as indicated in f. h Knockdown efficiencies of DNA helicases including eIF4A, Ded1, DHX29, RHA, and VASA were evaluated by real-time PCR. i. VHLα protein was examined in DNA helicase-deficient 293T cell by western blots. j Establishment of eIF4A-overexpressing (HA-eIF4A) and eIF4A-silencing (sheIF4A-1 and sheIF4A-2) cell lines was validated by real-time PCR. k VHLα protein alterations upon eIF4A overexpression and knockdown were evaluated by immunoblotting in 293T cells. l Relative VHLα proteins were examined in 293T in response to eIF4A-specific inhibitors for 0, 12, and 24 h (pateamine A, 15 μM; hippuristanol 5 μM; silvestrol 20 nM). m Consistent VHL transcript abundance in response to eIF4A inhibitors treatments as described in l. was measured by real-time PCR. n Representative IHC images of VHLα− and VHLα+ renal tumors (left) and analysis of relative expression of eIF2A and eIF4A regarding VHLα status by real-time PCR. Data are presented as mean ± S.D for three independent experiments. n.s., no significance; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.