Fig 2: Trp53 knockout partially restores platelet production in Rbm8aKOMK mice(A) Schematic diagram showing the generation of megakaryocyte-specific Rbm8a/Trp53 double-knockout mice (for the detail, see Figure S5A).(B) Bar graph shows platelet counts of 6- to 8-week-old mice of the indicated genotypes (mean ± SEM). N, sample number. Because most of Rbm8aKOMK-p53Homo mice died at 3–5 weeks of age, data were acquired from only three mice.(C) H&E staining of bone marrow sections from mice of indicated genotypes; magnified images of representative cells are shown.(D) Stacked-column bar graph shows the proportion (%) of megakaryocytes in bone marrow sections with different morphological features. All images were collected from three to five mice for each genotype.(E) Dot plots show the size of megakaryocytes measured from H&E staining (1.0 = 500 µm2); for each, 100–200 megakaryocytes from three to five mice of each genotype (mean ± SEM; ns, not statistically significant, ****p < 0.0001) that were measured.(F) IHC staining of Rbm8aKOMK-p53Het/p53Homo bone-marrow sections using anti-p21. Yellow arrow indicates megakaryocytes. The level of p21 was measured as in Figure 1B; ****p < 0.0001.(G) IHC was performed as in panel F using anti-phospho-H3. Red and black arrowheads indicate phospho-H3-positive and -negative megakaryocytes, respectively. The percentage of phospho-H3-positive cells was measured from three mice for each genotype (~100 megakaryocytes); **p < 0.01.

Fig 3: The synthetic lethal interaction between EJC and eIF4E is essential for cancer cell migration. (A) Representation of GO terms enriched in synthetic lethal partners of eIF4E in a cellular context. Each circle represents a gene, and the size of the circle corresponds to the -log10(p value) from a paired Wilcoxon test to show the significance of the mean difference of the phenotype between eIF4E+/- and eIF4E+/+ MEFs in the screen.(B) Cartoon of the exon-junction complex.(C) Schematic representation of the RNA-seq experiment.(D) Heatmap representation of differentially expressed genes in comparison to eIF4E or Rbm8a depletion. Although the alterations were minor when Rbm8a or eIF4E was depleted (left and middle columns, respectively), more significant changes were observed when Rbm8a was depleted in eIF4E+/- MEFs (right column). Each line represents the value calculated based on log2(FC) × -log10(p value).(E) Enriched GO terms of differentially expressed genes in eIF4E+/- Rbm8a KD versus eIF4E+/- Scramble MEFs (right column of Figure 4D). The size of the dots represents the number of genes in each category.(F) qPCR analysis of mRNA targets (Itga5, Mmp2, Snail1) that are specifically downregulated in eIF4E+/- Rbm8a KD. Multiple t tests were performed to determine statistical significance (n > 3).(G) Schematic representation of transwell migration assay and a representative confocal image of migrated cells. Migrated cells were counted for each group (n > 30). The bottom right graph represents the normalized value of Rbm8a KD over the scramble control in eIF4E+/+ and eIF4E+/- MEFs. Multiple t tests were performed to determine statistical significance.(H) Progression-free survival probability in liver cancer using TCGA data. The p value represents the interaction score between eIF4E and Rbm8a expression. A two-way ANOVA test was performed to determine statistical significance.All values represent the mean + SEM. *p < 0.01, **p < 0.001, ***p < 0.0001. See also Figure S4.

Fig 4: Y14 deficiency compromises megakaryocyte-maturation-related gene expression and impairs platelet function(A) The diagram shows the procedure of RNA-seq analysis in control or Y14-depleted HEL cells that were treated or untreated with PMA (see panel B).(B) HEL cells were transduced with lentivirus expressing shY14 (+) or control shRNA (-) for 48 h at a multiplicity of infection of 5, followed by mock-treated or PMA (5 nM) treatment for 3 days. RT-qPCR analysis of the indicated genes in HEL cells that were transfected with shRNA and then mock-treated or treated with PMA as in panel (A). The experiments were performed in triplicate (mean ± SEM; *p < 0.05, **p < 0.01).(C) Whole blood of Con and Rbm8aKOMK mice was collected and stained for IgG, GP-VI, GP-IX, a2bß3, and CD41 (Con: blue; Rbm8aKOMK: red). Platelets were gated by forward scattering/side scattering characteristics. Representative flow cytometry profiles showing the indicated glycoproteins or integrins.(D) Blood was mock-treated (resting; dashed lines) or treated with PAR4 agonist (solid lines) and subsequently stained with an antibody against JON/A (phycoerythrin), fibrinogen, vWF, or P-selectin. Flow cytometry profiles of platelet glycoproteins are shown as in panel (C).

Fig 5: p53 inhibition reverses the differentiation defects of Rbm8aKOMK megakaryocytes(A) IHC staining of Con and Rbm8aKOMK bone-marrow sections using antibodies against p53 and p21; their relative level was measured as in Figure 1B.(B) HEL cells were mock- or PMA-treated for 3 days. Immunoblotting was performed to examine Y14 and p53 proteins.(C) HEL cells were transduced with control or shY14-lentivirus, followed by PMA induction. Immunoblotting was performed as in panel (B).(D) HEL cells were treated as in panel C; immunoblotting was performed using antibodies against indicated proteins. A diagram shows that Y14 depletion increases the expression of p53 and 21 and decreases the expression of four cell-cycle regulators examined.(E) HEL cells were transduced as in panel C followed by mock (-) or PFT-a (1 or 5 µM) treatment. The relative levels of p21, Cdc20, and phospho-H3 (pH3) are indicated below the respective blots.(F) HEL cells were transduced with shY14-lentivirus and treated with PMA followed by mock or PFT-a treatment. DNA histogram analysis was performed as in Figure 2E.(G) The diagram depicts isolation and culture of megakaryocytes from E14.5 fetal liver. Isolated fetal liver stromal cells were cultured for 3 days in the presence of thrombopoietin (TPO). Megakaryocytes were enriched and cultured for another 3 days in the presence of TPO and then subjected to different analyses (panels H and I).(H) Giemsa staining of an enriched fraction containing megakaryocytes (yellow arrows). Bar graphs show the average size (µm2) of >350 cells (middle panel) and the percentage of CD41+ (right panel); experiments were performed in triplicate (mean ± SEM; **p < 0.01, ***p < 0.001).(I) Immunoblotting of the megakaryocyte-containing fraction was performed using antibodies against indicated proteins; residual Y14 signal was from nonmegakaryocytes in the coculture.(J) As in panel H, the megakaryocyte-containing fraction was mock- or PFT-a treated, followed by Giemsa staining or flow cytometry using anti-CD41. Bar graphs are shown as in panel F (mean ± SEM; *p < 0.05, **p < 0.01). Scale bars in panel G are the same as that in panel (F).(K) Immunoblotting was performed in HEL cells that were transfected with the empty (vec) or p53-expressing vector (lanes 1–2) or transduced with control (shC) or p53-targeting shRNA (shp53) lentivirus (lanes 3–4). A diagram shows that p53 suppresses Y14 expression.