Fig 2: Activation of eIF4E is responsible for both SP20H knockout and TNF-a stimulation induced B7-H3 translation. (A) Total protein expression of B7-H3, phospho-p38, and phospho-eIF4E in SB203580 treated SP20H knockout or TNF-a pretreated SK-OV-3 cells. (B) Changes of p38 MAPK-eIF4E signaling and B7-H3 expression in SK-OV-3 cells transduced with the indicated sgRNAs under TNF-a stimulation. (C) The expression levels of B7-H3 after interfering with eIF4E in the SP20H knockout SK-OV-3 cells. The values represent the mean ratios of B7-H3high/B7-H3low population of triplicate measurements±SD. (D) Schematic illustration of two ways to induce B7-H3 expression via the p38 MAPK- eIF4E signaling.

Fig 3: Analysis of the molecular pathways underlying GDF11-induced MSC differentiation. A, MSCsVector and MSCGDF11 were cultured in the presence or absence of VEGF165 for 14days. Total (T) and phosphorylated (P) ERK and EIF4E were examined by Western blot (n = 3). B, Quantitative analysis of A. The density of each band was calibrated with its corresponding Actin band. Relative phosphorylation levels were quantified by dividing P over T, which was then compared with the level in control MSCsVector to set as 1. C, MSCs were transfected with control (NC)- or GDF11-specific siRNA. Total (T) and phosphorylated (P) ERK and EIF4E were examined by Western blot (n = 3). D, Quantitative analysis of C in the same way as B. E, MSCsVector and MSCGDF11 were cultured with VEGF165 for EC differentiation in the presence or absence of inhibitors for TGF-ß receptor (LY2109761) or for ERK (SD5978) for 14days. T- and P- ERK and EIF4E were examined by Western blot. F, Quantitative analysis of E. Data are presented as the mean ± SD. *P < 0.05; **P < 0.01 and ***P < 0.001. Each WB was repeated for at least 2 times

Fig 4: Effect of GDF11 on the differentiation of MSCs into endothelial-like cells was blocked by ERK inhibitor. MSCVector and MSCGDF11 were treated with inhibitors for ERK (SD5978) or for TGF-ß receptor (LY2109761) or with DMSO as a control during the differentiation of MSCs into endothelial-like cells for 14 d. A, Representative results of flow cytometry analysis of MSCVector (Vector) and MSCGDF11 (GDF11) for their expression of CD31 and VEGFR2. B, Quantitative analysis of A (n = 3). C, Immunofluorescent staining of MSCVector and MSCGDF11 with antibody against CD31 (red) after culture with or without ERK inhibitor. Nucleus was stained with Hochest. Scale bars = 100 µm. D, Quantitative analysis of C (n = 3). CD31+ rate was obtained by dividing CD31+ cells (red) by total number of cells (blue) in a picture. E, Western blot analysis of proteins in MSCVector and MSCGDF11 in the presence of absence of inhibitors LY2109761 or SD5978during the differentiation of MSCs into endothelial-like cells. F, Quantitative analysis of E (n = 2). Each WB was repeated for at least 2 times. G, Schematic diagram of the proposed mechanisms by which GDF11 promotes MSC differentiation into endothelial-like cells. GDF11 interacts with TGF-ß receptor, resulting in activation of the ERK/EIF4E pathway, which enhances the expression of proteins for angiogenesis and anti-apoptosis, and augment of EC markers during cultured with VEGFA. Data are presented as the mean ± SD. *P < 0.05; **P < 0.01 and ***P < 0.001. BCL-2 for B-cell lymphoma-2 protein; BAX for Bcl2 Associated X Protein