| 名稱 | Brefeldin A |
| 描述 | Brefeldin A (Cyanein) belongs to the class of macrolide antibiotics and is an ATPase inhibitor (IC50=0.2 μM). Brefeldin A can induce tumor cell differentiation and apoptosis, and also possesses autophagy inhibitory activity. |
| 細(xì)胞實(shí)驗(yàn) | HF1A3, HF4.9 cell viability upon the treatments is tested using double staining of cells with YO-PRO 1/PI and SYTO16/PI probes. To access cell proliferation, cells are treated with 0–100 ng/mL Brefeldin A in complete medium for 20 hours before adding 1 μCi/mL [methyl-3H]-thymidine for additional 4 hours at 37 °C. The incorporated radioactive thymidine is quantified by scintillation counting with Microbeta counter. To examine long-term effects of Brefeldin A treatment, cells are seeded at initial concentration 105 cells/mL and treated with 0-75 ng/mL Brefeldin A for up to 5 days. At the time indicated, a sample of cells is removed and viable cell number is assessed by standard Trypan Blue exclusion assay.(Only for Reference) |
| 激酶實(shí)驗(yàn) | ELISA-based active site binding assay: Samples (lysed cells or tissue homogenates) are treated for 1 h at room temperature with the biotinylated active site probe PR-584 (5-15 μM). Samples are denatured by addition of SDS (0.9% final) and heating to 100 °C for 5 min. The denatured samples are transferred to a 96-well or 384-well filter plat, mixed with streptavidin-sepharose beads (2.5-5 μL packed beads/well), and incubated for 1 h at room temperature on a plate shaker. The beads are washed 5 times with 100-200 μL /well of ELISA buffer (PBS, 1% bovine serum albumin, 0.1% Tween-20) by vacuum filtration. The beads are incubated overnight at 4 °C on a plate shaker with the following antibodies recognizing the six catalytic subunits diluted into ELISA buffer: β5, β1, and β2 diluted 1:3000, LMP7 and LMP2 diluted 1:5000, and MECL-1 diluted 1:1000. The beads are washed 5 times with 100-200 μL /well of ELISA buffer and incubated with HRP-conjugated secondary antibody diluted 1:5000 in ELISA buffer and incubated 2 h at room temperature on a plate shaker. The beads are washed 5 times with 100-200 μL /well of ELISA buffer and developed for chemiluminsecence signal using the supersignal ELISA pico substrate following the manufacturer's instructions. Luminescence is measured on a plate reader and converted to ng of proteasome or μg/ml of lysate by comparison with 20S proteasome or untreated cell lysate standard curves. For proteasome inhibitor studies, active site probe binding values are expressed as the percent of binding relative to DMSO treated cells. |
| 體外活性 | 方法：腫瘤細(xì)胞 HL60 、K562 和 HT-29 用 Brefeldin A (2 μM) 處理 72 h，使用 DNA filter elution assay 檢測(cè) DNA 片段。
結(jié)果：Brefeldin A 以不同的動(dòng)力學(xué)誘導(dǎo) DNA 斷裂。HL60 細(xì)胞在 15 h 內(nèi)觀察到完整的 DNA 片段，而 K562 和 HT-29 細(xì)胞則需要 48-72 h。[1]
方法：人乳腺癌細(xì)胞 MDA-MB-231 用 Brefeldin A (0.05-1 μg/mL) 處理 24 h，使用 Western Blot 方法檢測(cè)靶點(diǎn)蛋白表達(dá)水平。
結(jié)果：PARP 切割是細(xì)胞死亡的標(biāo)志性事件，可以在 Brefeldin A 處理的懸浮 MDA-MB-231 細(xì)胞中檢測(cè)到。[2] |
| 體內(nèi)活性 | 方法：為檢測(cè)體內(nèi)抗腫瘤活性，將 Brefeldin A (15 mg/kg in 10% ricinus oil+5% DMSO+10% ethanol+75% physiologic saline) 腹腔注射給攜帶人結(jié)直腸癌腫瘤 HCT116 的 BALB/c 小鼠，每天一次，持續(xù)四周。
結(jié)果：Brefeldin A 在體內(nèi)抑制 HCT116 腫瘤生長。[3]
方法：為檢測(cè)體內(nèi)抗腫瘤活性，將 Brefeldin A (16-64 mg/kg in distilled water containing 0.05% Tween 80) 腹腔注射給攜帶腫瘤 LOX IMVI 的 Athymic NCr nu/nu 小鼠，每天兩次，持續(xù)五天。
結(jié)果：Brefeldin A 在體內(nèi)顯示出抗腫瘤活性，小鼠壽命增加 65%-100%，第 60 天幸存者增加 17%-50%。[4] |
| 存儲(chǔ)條件 | Store at low temperature,Keep away from moisture,Store under nitrogen
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 | Ethanol : 2.81 mg/mL (10.02 mM), Sonication is recommended.
10% DMSO+40% PEG300+5% Tween 80+45% Saline : 4 mg/mL (14.27 mM), Sonication is recommended.
DMSO : 128.75 mg/mL (459.23 mM), Sonication is recommended.
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| 關(guān)鍵字 | Mitophagy | Mitochondrial Autophagy | Inhibitor | inhibit | HSV | Herpes simplex virus | CRISPR/Cas9 | CRISPR | Cas9 | Brefeldin A | Autophagy | ATPase (HCT 116) | Antibiotic |
| 相關(guān)產(chǎn)品 | Guanidine hydrochloride | Aceglutamide | Neomycin sulfate | Terbinafine hydrochloride | Hemin | Hydroxychloroquine | Dimethyl sulfoxide | Sodium bicarbonate | Hyaluronic acid sodium (MW 20 kDa) | Sodium diacetate | Sulfamethoxazole sodium | D(+)-Raffinose pentahydrate |
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