Stattic (STAT3 Inhibitor V) is a STAT3 inhibitor (IC50=5.1 μM) that selectively inhibits STAT3 activation, dimerization, and nuclear translocation. Stattic has antitumor activity and induces apoptosis.

STAT3:5.1 μM (cell free)

方法：人胰腺癌细胞 PANC-1 和 BxPc-3 用 Stattic (1-10 μM) 处理 12-48 h，使用 CCK-8 方法检测细胞活力。
结果：Stattic 以浓度和时间依赖的方式降低 PANC-1 和 BxPc-3 细胞增殖。Stattic 处理 24 h 后对 BxPc-3 和 PANC-1 细胞的 IC50 分别为 3.135-5.296 μM 和 3.835-4.165 μM。[1]
方法：人肝癌细胞 HepG2 用 Stattic (5-20 μM) 处理 1 h，随后用 IL-6 或 IFN-γ刺激，使用 Western Blot 方法检测靶点蛋白表达水平。
结果：Stattic 的预孵育导致 STAT3 Tyr705 的磷酸化选择性降低，而 STAT1 Tyr701 的激活保持不变。[2]

方法：为检测体内抗肿瘤活性，将 Stattic (10 mg/kg) 腹腔注射给携带人胰腺癌肿瘤 PANC-1 的 BALB/c nude 小鼠，每天一次，持续四周。
结果：Stattic 通过灭活 STAT3 来抑制裸鼠肿瘤模型中的 PC 生长。[1]
方法：为研究在急性肝损伤中的作用，将 Stattic (5 mg/kg in DMSO:olive oil = 1:19) 单次腹腔注射给  LPS/d-GalN 诱导急性肝损伤的 BALB/c 小鼠。
结果：Stattic 对 LPS/d-GalN 诱导的肝损伤具有保护作用，其保护作用可能与其抗炎和抗凋亡作用有关。[3]

The screening was performed at approximately 30C. The specificity of screening hits was validated in analogous assays for binding of the test compounds to the SH2 domains of STAT1, STAT5, and Lck. The final concentration of buffer components used for all FP assays was 10 mM HEPES (pH 7.5), 1 mM EDTA, 0.1% Nonidet P-40, 50 mM NaCl, and 10% DMSO. The absence of dithiothreitol is essential for inhibitory activity. The sequences of the peptides were: STAT3, 5-carboxyfluorescein-GY(PO3H2)LPQTV-NH2; STAT1, 5-carboxyfluorescein-GY(PO3H2)DKPHVL; STAT5, 5-carboxyfluorescein-GY (PO3H2)LVLDKW; and Lck, 5-carboxyfluorescein-GY(PO3H2)EEIP. Peptides were >95% pure. For specificity analysis at 30°C, proteins were used at 150 nM (STAT1, STAT3, and STAT5). For specificity analysis at 37°C, proteins were used at 370 nM (STAT3) or 100 nM (Lck). Proteins were incubated with test compounds in tubes at the indicated temperatures for 60 min prior addition of the respective 5-carboxyfluorescein labeled peptides (final concentration: 10 nM). Analysis of c-Myc/Max and Jun/Jun dimerization and DNA binding at 37°C was performed as described but in the absence of DTT. Before measurement at room temperature, the mixtures were allowed to equilibrate for at least 30 min. Test compounds were used at the indicated concentrations diluted from 20× stock in DMSO. Binding curves and inhibition curves were fitted with SigmaPlot. All competition curves were repeated three times in independent experiments. For the analysis of time dependence of the inhibition, the components were mixed from stock solutions kept at 0C and then incubated at 37C. Aliquots were taken at the indicated time points [1].

MDA-MB-231, MDA-MB-435S, and MDA-MB-453 cells were seeded. at 5 × 10^4 cells in 6-well plates, grown for 24 hr before adding DMSO or Stattic (final DMSO concentration 0.1%) and then incubated with the inhibitor for 24 hr. All cells were collected and resuspended in buffer (0.1% sodium citrate, 0.1% Triton X-100, 20 μM propidium iodide) and incubated for 3 hr before 10^4 cells per sample were analyzed by flow cytometry with a FACSCalibur equipped with a 488 nm laser [1].