Fig. 4: Downregulation of eIF4A1/2 affects the perichromosomal layer. | Nature Communications

Fig. 4: Downregulation of eIF4A1/2 affects the perichromosomal layer.

From: The DEAD-box helicase eIF4A1/2 acts as RNA chaperone during mitotic exit enabling chromatin decondensation

Fig. 4

A Time-lapse images of HeLa cells stably expressing H2B-mPlum and EGFP-CCDC137, transfected with a combination of 20 nM eIF4A1 and eIF4A2 siRNA oligos for 72 h showing cytosolic EGFP-CCDC137 aggregates (red arrowhead) seen in 3 independent experiments. Time is normalized to the first anaphase frame. B High-resolution imaging of fixed HeLa cells stably expressing H2B-mPlum (magenta in overlay) and EGFP-CCDC137 (green in overlay), transfected with 20 nM control, a combination of eIF4A1 and eIF4A2 or Ki-67 siRNA oligos for 72 h showing cytosolic EGFP-CCDC137 aggregates (red arrowheads). Line scans presented in (C) are indicated. C Line scans of the EGFP-CCDC137 fluorescence intensity in the cells shown in (B). Line scans are drawn across 200 pixels in each cell, and the EGFP-CCDC137 signal is presented in gray values. D Quantitation of EGFP-CCDC137 localization from one experiment as in (B) showing cell percentage with more than 90 cells per condition. E Time-lapse images of HeLa cells expressing EGFP-Ki-67 transfected with 20 nM control or a combination of eIF4A1 and eIF4A2 siRNA oligos for 72 h and treated with 250 nM SiR-DNA for 2 h to visualize chromatin. Time is normalized to the first anaphase frame. Red arrowheads indicate unsegregated chromatin masses seen in 5 independent experiments. F Ki-67 immunostaining (green in overlay) of HeLa cells stably expressing H2B-mCherry (magenta in overlay) transfected with 20 nM control or a combination of eIF4A1 and eIF4A2 siRNA oligos for 72 h at different stages of mitotic exit. G Quantitation of the Ki-67 mean fluorescence intensity in anaphase and telophase from three independent experiments as in F. The violin plots show the means of three independent experiments (triangles) each including 18 (siCtrl) and 10 (sieIF4A1/2) cells for early/late anaphase and 22 (siCtrl) and 15 (sieIF4A1/2) cells for late telophase and the overall mean ± s.e.m. Significance was tested by a two-tailed unpaired t test with Welch’s correction (early/late anaphase, ***P = 1.35 × 10-5; early telophase, ***P = 7.95 × 10−5). Scale bars: 10 µm. Source data are provided as a Source Data file.

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