Figure 2. MB-associated DDX3X mutations impair mRNA translation.

(a) ³⁵S-Met/Cys labeling of nascent polypeptides in HEK293T cells transfected for 36 h with the indicated FLAG-tagged DDX3X variants or empty vector (control). Values are the mean counts per minute (CPM) of a minimum of three replicates per sample, normalized to total protein level as a function of labeling time (min) with ³⁵S-Met/Cys. Error bars represent SEM at each time point. Two-way ANOVA: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. Western blot (right) shows DDX3X levels in pooled replicates at each time point; β-actin served as the loading control. (b) Puromycin incorporation analysis in HEK293T cells expressing EGFP-tagged wild-type or cancer-related mutant DDX3X (G302V, G325E, M370R). Puromycin incorporation was conducted as indicated in Material and Methods. Translation is monitored by staining against puromycin (red), while SGs are detected by staining against eIF4G (magenta). Green color represents EGFP-tagged DDX3X, nuclei are stained with DAPI. Dashed lines denote cells with impaired translation as monitored by lack of puromycin; yellow boxes show magnified area at the right.