Suppression of the PI3K Subunit p85α Delays Embryoid Body Development and Inhibits Cell Adhesion
Suppression of the PI3K Subunit p85α Delays Embryoid Body Development and Inhibits Cell Adhesion
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DOI：
10.1002/jcb.23285
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发表时间：
2011-12-01
影响因子：
4
通讯作者：
Schwanbeck, Ralf
中科院分区：
文献类型：
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作者：
Gurney, Susan M. R.;Forster, Peter;Schwanbeck, Ralf
磷脂酰肌醇-3-激酶（PI 3 Ks）在真核生物中发挥多种信号传导功能。我们使用小干扰RNA（Pik 3r 1 siRNA）抑制PI 3 K调节亚基p85 α，并在悬滴培养中检查对胚状体（EB）发育的影响。与阴性对照相比，我们观察到24小时内处理的EB的体积增加了150%。荧光激活细胞分选（FACS）分析显示，这种体积增加不是由于细胞增殖增加。相反，体积的增加似乎是由于细胞聚集和粘附减少。我们的观察结果进一步表明，40%的经处理的EB形成双EB而不是单EB，并且它们在铺板时粘附于培养皿的能力显著降低。在第一个96小时的时间过程表明，受损的坚持是短暂的，并解释了最初的12小时延迟EB发展。定量PCR表达分析表明，粘附分子整合素β 1（ITGB 1）是瞬时下调的p85 α抑制。总之，我们发现，抑制p85 α导致延迟形成紧凑的EB，伴随着短暂的EB无法进行正常的细胞-细胞和细胞-基质粘附。J.细胞。112：3573-3581，2011中。(C)2011 Wiley Periodicals，Inc.
Phosphatidylinositol-3-kinases (PI3Ks) exert a variety of signaling functions in eukaryotes. We suppressed the PI3K regulatory subunit p85 alpha using a small interfering RNA (Pik3r1 siRNA) and examined the effects on embryoid body (EB) development in hanging drop culture. We observed a 150% increase in the volume of the treated EBs within 24 h, compared to the negative controls. Fluorescence Activated Cell Sorting (FACS) assays showed that this increase in volume is not due to increased cellular proliferation. Instead, the increase in volume appears to be due to reduced cellular aggregation and adherence. This is further shown by our observation that 40% of treated EBs form twin instead of single EBs, and that they have a significantly reduced ability to adhere to culture dishes when plated. A time course over the first 96 h reveals that the impaired adherence is transient and explained by an initial 12-hour delay in EB development. Quantitative PCR expression analysis suggests that the adhesion molecule integrin-beta 1 (ITGB1) is transiently downregulated by the p85 alpha suppression. In conclusion we found that suppressing p85 alpha leads to a delay in forming compact EBs, accompanied by a transient inability of the EBs to undergo normal cell-cell and cell-substrate adhesion. J. Cell. Biochem. 112: 3573-3581, 2011. (C) 2011 Wiley Periodicals, Inc.