| 名稱 | Thiazovivin |
| 描述 | Thiazovivin, a ROCK inhibitor (IC50: 0.5 μM), increases the survival rate of hESC. |
| 細(xì)胞實(shí)驗(yàn) | Chemically defined and feeder-free human embryonic stem cell (hESC) culture was described briefly as following. hESCs were grown on Matrigel-coated tissue culture plates in N2B27-CDM [DMEM-F12 supplemented with 1× N2 supplements, 1× B27 supplements, 2 mM L glutamine, 0.11 mM 2-mercaptoethanol, 1× nonessential amino acids, and 0.5 mg/mL BSA (fraction V)] and 20 ng/mL bFGF. Human ESCs were passaged every five to six days with 0.05% trypsin. Murine ESCs are cultured in knockout DMEM supplement with 2 mM L glutamine, 1× nonessential amino acids, 15% serum replacement, and 1 × 10^3 ng/mL leukemia inhibitory factor (LIF). For clonal survival assays, single hESCs were diluted to clonal density and plated onto 96-well Matrigel-coated plate. For low-density survival assays, 500 cells were plated onto 96-well Matrigel-coated plate. To visualize hESC colonies, cultures were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 5 min, washed once in PBS, then stained for alkaline phosphatase (ALP) activity as described in the manufacturer's instructions. ALP-positive colonies were counted on an inverted microscope. For growing hESCs in mouse medium, HES2, HUES7, HUES9, and HUES1-Oct4-GFP were cultured in murine ESC (mESC) growth media supplemented with 1-μM mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor PD0325901 and 5-μM p38 inhibitor SB202190 and 1 × 10^3 human LIF [1]. |
| 激酶實(shí)驗(yàn) | Synthesis of compound immobilized affinity matrixes: Compounds Tzv and its inactive analog (10 mg each) in DMSO (500 μL) and Et3N (10.4 μL) were added to Reacti-Gel (0.5 mL, 25 μmol) that was washed by DMSO in an Eppendorf vial. The reaction mixture was incubated at room temperature until the starting material disappeared (determined by HPLC). After the disappearance of the starting material, ethanolamine (15 μL) was added and the resulting mixture was incubated at room temperature overnight to block the Reacti-Gel. The resulting affinity matrices were washed thoroughly with DMSO (500 μL × 4), PBS (500 μL × 2), and stored at 4 °C in NaN3 solution (0.1% in PBS). Affinity pull-down was performed as described previously. Briefly, whole cell lysates were pretreated with the unfunctionalized affinity matrix at 4 °C for 1.5 h. After washing three times, samples were incubated with the positive or negative affinity matrix at 4 °C for 1 h. After heat shock, samples were loaded and separated on a 4–20% Tris-Glycine SDS PAGE and silver stained with a Silver Stain Plus Kit. The differentially retained protein bands were cut, destained, and analyzed with LCMS [1]. |
| 體外活性 | 當(dāng)細(xì)胞在E-cadherin阻斷抗體的存在下培養(yǎng)時(shí)，細(xì)胞在Thiazovivin處理后形成的大型、緊密的聚集體嚴(yán)重受阻，且觀察到廣泛的細(xì)胞死亡。Thiazovivin（2 μM）抑制ROCK活性，并在與Y-27632（10 μM）相似的水平上保護(hù)hESCs，后者是一種廣泛使用的選擇性ROCK抑制劑[1]。在Thiazovivin處理組中，囊胚和胚胎細(xì)胞團(tuán)在給養(yǎng)細(xì)胞上的附著率高于對(duì)照組。Thiazovivin處理增加了OCT4和NANOG基因的多能性標(biāo)記以及粘附分子E-cadherin[2]。通過(guò)在重編程培養(yǎng)中添加Thiazovivin，CB細(xì)胞的重編程效率增加了10倍以上[3]。 |
| 存儲(chǔ)條件 | Pure form: -20°C for 3 years | In solvent: -80°C for 1 year
Shipping with blue ice/Shipping at ambient temperature. |
| 溶解度 | DMSO : 14 mg/mL (44.96 mM), Sonication is recommended.
10% DMSO+40% PEG300+5% Tween 80+45% Saline : 2 mg/mL (6.42 mM), Sonication is recommended.
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| 關(guān)鍵字 | Thiazovivin | ROK | ROCK | Rho-kinase | Rho-associated protein kinase | Rho-associated kinase | Inhibitor | inhibit |
| 相關(guān)產(chǎn)品 | Y-27632 dihydrochloride | ITX3 | CID-5056270 | Fasudil hydrochloride | ROCK-IN-5 | Fasudil | Tofacitinib | CAY10746 | Y-27632 | RKI-1447 | SAR407899 hydrochloride | GSK180736A |
| 相關(guān)庫(kù) | 抑制劑庫(kù) | 經(jīng)典已知活性庫(kù) | 抗癌化合物庫(kù) | 已知活性化合物庫(kù) | 激酶抑制劑庫(kù) | 神經(jīng)再生化合物庫(kù) | 細(xì)胞骨架化合物庫(kù) | NO PAINS 化合物庫(kù) | TGF-β/Smad靶點(diǎn)化合物庫(kù) | 膜蛋白靶向化合物庫(kù) | 抗代謝疾病化合物庫(kù) | 細(xì)胞重編程化合物庫(kù) |