Pdcd4 deficiency reduces the formation of TIA-1⁺ SGs in ox-LDL-treated macrophages.

<p>(A, B) Primary macrophages from WT (A) or <i>Pdcd4</i>^-/- (B) mice (n≥3 per group) were stimulated with ox-LDL (50 μg/ml) for 24 h, the formation of SGs was examined by indirect immunofluorescence. TIA-1 (red); nuclei (blue). The original magnification is 630. Scale bar = 10 μm. (C) Positive percentage of cells containing TIA-1⁺ SGs. Data are presented as mean ± s.e.m. ***<i>P</i> <0.001.</p

AKT-eIF2α axis is involved in the regulation of Pdcd4 on the formation of ox-LDL or HFD-induced SGs.

(A, B) Western-blot assay of p-eIF2α in HeLa cell transfected with pEGFP-C1 (Mock) or pEGFP-C1-Pdcd4 (Pdcd4) in the presence or absence of ox-LDL (50 μg/ml). Representative (A) and statistic (B) data are shown. (C-E) Primary macrophages from WT and Pdcd4-/- mice (n≥4 per group) were treated with ox-LDL (50 μg/ml) for 24 h. Representative western-blot (C) and statistic data of p-eIF2α and p-AKT (D, E) are shown. (F, G) Primary macrophages from Pdcd4-/- mice (n = 4–6) were treated with ox-LDL (50 μg/ml) for 24 h in the presence or absence of MK2206 (2 μM). The expression of p-eIF2α and p-AKT were determined by western-blot. (H-J) Representative (H) and statistic (I, J) western-blot analysis of p-eIF2α and p-AKT in macrophages from WT and Pdcd4-/- mice (n = 4 per group) fed on ND or HFD. Data are presented as mean ± s.e.m. *P P P <0.001.</p

Pdcd4 deficiency reduces the SG formation in macrophages and liver tissues from HFD-fed mice.

<p>(A, B) The formation of TIA-1⁺ SGs was examined in primary macrophages from WT and <i>Pdcd4</i>^<i>-/-</i> mice (n = 2–4 per group) fed on ND or HFD by immunofluorescence. Representative (A) and statistical (B) data are shown. *<i>P</i> <0.05. (C) The formation of TIA-1⁺ SGs was examined in liver tissues from WT and <i>Pdcd4</i>^<i>-/-</i> mice (n = 4 per group) fed on ND or HFD by immunofluorescence. TIA-1 (red); nuclei (blue). The original magnification is 1000 (A) or 630 (C). Scale bar = 10μm.</p

Pdcd4 participates in the SG formation through its RNA-binding region.

<p>(A) Structural models showing the location of two RNA-binding motifs (RBM1 and RBM2) in WT <i>Pdcd4</i> genes, and three different truncated <i>Pdcd4</i> genes depleted of RBM1, RBM2, or both, respectively. (B) Representative western-blot for ectopic expression of GFP-Pdcd4 in HepG2 cells transfected with various truncated plasmids. (C) HepG2 cells were transfected with various truncated plasmids and then were stimulated with ox-LDL (50 μg/ml) for 16 h, the SG formation was examined by immunofluorescence. Exogenous Pdcd4 is visualized with GFP (green), TIA-1 was stained with rhodamine (red). Cells were counterstained with DAPI (blue). Arrow head indicates SGs in cells. The original magnification is 1000. Scale bar = 20μm. Data are from three independent experiments. (D) Number of SGs in HepG2 cells transfected with various truncated plasmids. Data are presented as mean ± s.e.m. ***<i>P</i> <0.001.</p

A working model shows the roles of Pdcd4 in the formation of SGs induced by ox-LDL or HFD.

<p>Pdcd4 serves as a key component of ox-LDL-induced SGs through its RNA-binding activity. On the other hand, Pdcd4 can enhance eIF2α phosphorylation by attenuating AKT activation, thereby promoting the formation of SGs.</p

Pdcd4 is co-localized with specific markers of SGs in ox-LDL-treated macrophages.

<p>Primary macrophages from WT mice (n = 6) were stimulated with ox-LDL (50 μg/ml) for 24 h. The expression and co-localization of Pdcd4 and SG markers were examined by immunofluorescence. Pdcd4 immunoreactivity was visualized with FITC (green), TIA-1, FXR1 and eIF4A were detected with rhodamine (red). Cell nuclei were stained with DAPI (blue). The original magnification is 630. Scale bar = 10 μm.</p

Ectopic Pdcd4 is co-localized with specific markers of SGs in ox-LDL-treated HeLa cells.

<p>HeLa cells were transfected with <i>pEGFP-C1-Pdcd4</i> in the presence or absence of ox-LDL (50 μg/ml) for 24 h. Intracellular localization of exogenous Pdcd4 was visualized with GFP (green). SG markers (TIA-1, FXR1 and eIF4A) were examined by rhodamine (red) using immunofluorescence. Cells were counterstained with DAPI (blue). The original magnification is 1000. Scale bar = 20 μm. Representative (A) and statistical (B) data are shown. Data represent more than three independent experiments with similar results. ***<i>P</i> <0.001.</p

Blastocyst-Inspired Hydrogels to Maintain Undifferentiation of Mouse Embryonic Stem Cells

Stem cell fate is determined by specific niches that provide multiple physical, chemical, and biological cues. However, the hierarchy or cascade of impact of these cues remains elusive due to their spatiotemporal complexity. Here, anisotropic silk protein nanofiber-based hydrogels with suitable cell adhesion capacity are developed to mimic the physical microenvironment inside the blastocele. The hydrogels enable mouse embryonic stem cells (mESCs) to maintain stemness in vitro in the absence of both leukemia inhibitory factor (LIF) and mouse embryonic fibroblasts (MEFs), two critical factors in the standard protocol for mESC maintenance. The mESCs on hydrogels can achieve superior pluripotency, genetic stability, developmental capacity, and germline transmission to those cultured with the standard protocol. Such biomaterials establish an improved dynamic niche through stimulating the secretion of autocrine factors and are sufficient to maintain the pluripotency and propagation of ESCs. The mESCs on hydrogels are distinct in their expression profiles and more resemble ESCs in vivo. The physical cues can thus initiate a self-sustaining stemness-maintaining program. In addition to providing a relatively simple and low-cost option for expansion and utility of ESCs in biological research and therapeutic applications, this biomimetic material helps gain more insights into the underpinnings of early mammalian embryogenesis