Fig. 1: eIF4A1/2 is important for in vitro chromatin decondensation.
A Mitotic chromatin clusters were decondensed in control‐ (Mock) and eIF4A1/2‐depleted (ΔeIF4A1/2) Xenopus egg extracts supplemented with buffer or recombinant Xenopus eIF4A1, eIF4A2 or eIF4A3. Samples were fixed after 2 h with 4% PFA and 0.5% glutaraldehyde, stained with DAPI, and analyzed by confocal microscopy. B For decondensation quantification, the boundary smoothness of the chromatin was analyzed. Means of two independent experiments (triangles), overall mean ± s.e.m. of in total more than 50 chromatin substrates for each condition as in (A). At some points, errors might be too small to be visible. Two-tailed unpaired Mann-Whitney U test (ΔeIF4A1/2, ***P = 3.3 × 10−18; +eIF4A3, ***P = 2.63 × 10−18). C Western blot analysis with a Xenopus eIF4A1/2 antibody of Mock‐ and eIF4A1/2‐depleted extracts, without or with the addition of recombinant Xenopus eIF4A1, eIF4A2 or eIF4A3. Nup62 is used as a loading control. D Mitotic chromatin clusters were decondensed and analyzed as in (A) but supplemented with buffer or recombinant wild-type eIF4A1, the ATPase deficient E183Q, or the RNA-binding deficient R362/365Q mutant. E The violin plot of the chromatin border smoothness. Means of three independent experiments (triangles), overall mean ± s.e.m. of in total more than 50 chromatin substrates for each condition as in (D). At some points, errors might be too small to be visible. Two-tailed unpaired Mann-Whitney U test (ΔeIF4A1/2, ***P = 7.65 × 10−19; + eIF4A1 R362/365Q, ***P = 4.96 × 10−19). F Western blot analysis with a Xenopus eIF4A1/2 antibody of Mock-, eIF4A1/2‐depleted, or eIF4A1/2‐depleted extracts supplemented with the indicated eIF4A1 proteins. Nup62 is used as a loading control. G Mitotic chromatin clusters were decondensed in Xenopus egg extracts in the absence or presence of 0.05 mg/ml RNA supplemented with buffer, 5 or 15 µM recombinant Xenopus eIF4A1 or 50 µg/ml RNase A and analyzed as in (A). H Violin plot shows the means of three independent experiments (triangles) and the overall mean ± s.e.m., of in total more than 40 chromatin substrates for each condition as in (E). At some points, errors might be too small to be visible. Two-tailed unpaired Mann-Whitney U test ( + RNA, ***P = 1.63 × 10−18; + RNA + eIF4A1 5 µM, ***P = 1.63 × 10−18). Source data are provided as a Source Data file. Scale bars: 10 µm.
