Fig. 2. EIF4A3 localizes in transcriptionally active nucleoli and safeguards the nucleolar structure.

(A) Representative IF images of eIF4A3 localization in U2OS cells left either untreated or stimulated with the ribosomal stress facilitators ActD^L and BMH-21. Insets depict magnifications of the regions designated in squares. Scale bar, 5 μm. DMSO, dimethyl sulfoxide. (B) Violin plot of high-content microscopy–based eIF4A3 nucleolar signal quantification under the experimental conditions described in (A). Nucleolar signal was calculated as the mean integrated intensity of endogenous eIF4A3 colocalizing with the nucleolar marker FBL. A total of 1000 to 2000 cells were analyzed per experiment (data are shown as means ± SD; n = 3 biological replicates; ****P < 0.001). a.u., arbitrary units. (C) Representative IF images of nucleolar structure in U2OS left untreated or treated with sieIF4A3, ActD^L, or their combination. FBL and UBF were used as fibrillar center and dense fibrillar component markers, respectively. Insets depict magnifications of the regions designated in squares. Scale bar, 5 μm. (D) Representative IF images of the nucleolus under the same experimental conditions (A) using nucleophosmin 1 (NPM1) as a granular component (GC) marker. Scale bar, 5 μm. (E) Electron microscopy (EM) images showing the impact of sieIF4A3 ± ActD^L on nucleolar morphology of U2OS cells. Dashed borders designate nucleoli. Scale bar, 2 μm. (F) Detection of eIF4A3 protein levels with immunocytochemistry in samples from patients with normal brain or glioblastoma. Regions in yellow squares are presented magnified in the bottom panel. Scale bar, 100 μm. (G) Same methodology in normal cervix or cervical squamous cell carcinoma specimens.