Puromycin aminonucleoside (NSC 3056) is the aminonucleoside portion of the antibiotic puromycin, and used in nephrosis animal models^[1]. Puromycin aminonucleoside induces apoptosis^[2]. Puromycin aminonucleoside is a reversible inhibitor of dipeptidyl peptidase II and cytosol alanyl aminopeptidase^[3].

Puromycin aminonucleoside (NSC 3056) (30 μg/mL) markedly increases p53 protein levels in podocytes. Puromycin aminonucleoside (NSC 3056)-induced podocyte apoptosis is p53 dependent. Puromycin aminonucleoside (NSC 3056) induces podocyte apoptosis in a time-dependent manner^[2]. The IC₅₀ values for PMAT-expressing and vector-transfected cells are 48.9 and 122.1 μM, respectively, suggesting expression of PMAT-enhanced cell sensitivity to Puromycin aminonucleoside. Puromycin aminonucleoside (NSC 3056) (250 μM) is toxic to both PMAT-expressing and vector-transfected cells. Puromycin aminonucleoside (NSC 3056) uptake in PMAT-expressing cells is fourfold higher at pH 6.6 than that at pH 7.4^[4].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

The number of podocytes per glomerulus is 95.5±17.6 in the control rats, 90.7 on Day 4 in Puromycin aminonucleoside (NSC 3056) (8 mg/100 g, i.v.)-treated nephrosis rats. The amount of nephrin per glomerulus in control rats is 1.02±0.11 fmol and those in Puromycin aminonucleoside (NSC 3056) nephrosis rats are reduced to 0.46±0.06 fmol and 0.35±0.04 fmol on Day 4 and Day 7. The nephrin amount per podocyte is significantly decreased association with the development of proteinuria in Puromycin aminonucleoside (NSC 3056) nephrosis rats^[5]. Rats given Puromycin aminonucleoside (NSC 3056) (100 mg/kg, s.c.) gain less weight and their serum creatinine levels are higher than the control rats^[6].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

294.31

C₁₂H₁₈N₆O₃

58-60-6

氨基核苷嘌呤霉素；嘌呤霉素氨基核苷

Room temperature in continental US; may vary elsewhere.

H₂O : 40 mg/mL (135.91 mM; ultrasonic and warming and heat to 50°C)

DMSO : 25 mg/mL (84.94 mM; ultrasonic and warming and heat to 60°C)

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80°C, 6 months; -20°C, 1 month。-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照 In Vitro 方式配制澄清的储备液，再依次添加助溶剂：

——为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂： PBS

  Solubility: 12.5 mg/mL (42.47 mM); Clear solution; Need ultrasonic
• 2.

  请依序添加每种溶剂： 5% DMSO    40% PEG300    5% Tween-80    50% saline

  Solubility: ≥ 2.5 mg/mL (8.49 mM); Clear solution
• 3.

  请依序添加每种溶剂： 5% DMSO    95% (20% SBE-β-CD in saline)

  Solubility: ≥ 2.5 mg/mL (8.49 mM); Clear solution
• 4.

  请依序添加每种溶剂： 10% DMSO    40% PEG300    5% Tween-80    45% saline

  Solubility: ≥ 2.08 mg/mL (7.07 mM); Clear solution

  此方案可获得 ≥ 2.08 mg/mL (7.07 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 20.8 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH₂O 中，得到澄清透明的生理盐水溶液

• 5.

  请依序添加每种溶剂： 10% DMSO    40% PEG300    5% Tween-80    45% saline

  Solubility: ≥ 2.08 mg/mL (7.07 mM); Clear solution

  此方案可获得 ≥ 2.08 mg/mL (7.07 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 20.8 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH₂O 中，得到澄清透明的生理盐水溶液

• 6.

  请依序添加每种溶剂： 10% DMSO    40% PEG300    5% Tween-80    45% saline

  Solubility: ≥ 2.08 mg/mL (7.07 mM); Clear solution

  此方案可获得 ≥ 2.08 mg/mL (7.07 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 20.8 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH₂O 中，得到澄清透明的生理盐水溶液

• 7.

  请依序添加每种溶剂： 10% DMSO    90% (20% SBE-β-CD in saline)

  Solubility: ≥ 2.08 mg/mL (7.07 mM); Clear solution

  此方案可获得 ≥ 2.08 mg/mL (7.07 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 20.8 mg/mL 的澄清 DMSO 储备液加到 900 μL 20% 的 SBE-β-CD 生理盐水水溶液中，混合均匀。

  将 2 g 磺丁基醚 β-环糊精加入 5 mL 生理盐水中，再用生理盐水定容至 10 mL，完全溶解，澄清透明
• 8.

  请依序添加每种溶剂： 10% DMSO    90% (20% SBE-β-CD in saline)

  Solubility: ≥ 2.08 mg/mL (7.07 mM); Clear solution

  此方案可获得 ≥ 2.08 mg/mL (7.07 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 20.8 mg/mL 的澄清 DMSO 储备液加到 900 μL 20% 的 SBE-β-CD 生理盐水水溶液中，混合均匀。

  将 2 g 磺丁基醚 β-环糊精加入 5 mL 生理盐水中，再用生理盐水定容至 10 mL，完全溶解，澄清透明
• 9.

  请依序添加每种溶剂： 10% DMSO    90% corn oil

  Solubility: ≥ 2.08 mg/mL (7.07 mM); Clear solution

  此方案可获得 ≥ 2.08 mg/mL (7.07 mM，饱和度未知) 的澄清溶液，此方案不适用于实验周期在半个月以上的实验。

  以 1 mL 工作液为例，取 100 μL 20.8 mg/mL 的澄清 DMSO 储备液加到 900 μL玉米油中，混合均匀。

• 10.

  请依序添加每种溶剂： 10% DMSO    90% corn oil

  Solubility: ≥ 2.08 mg/mL (7.07 mM); Clear solution

  此方案可获得 ≥ 2.08 mg/mL (7.07 mM，饱和度未知) 的澄清溶液，此方案不适用于实验周期在半个月以上的实验。

  以 1 mL 工作液为例，取 100 μL 20.8 mg/mL 的澄清 DMSO 储备液加到 900 μL玉米油中，混合均匀。

• [1]. Lacalle RA, et al. Cloning of the complete biosynthetic gene cluster for an aminonucleoside antibiotic, puromycin, and its regulated expression in heterologous hosts. EMBO J. 1992 Feb;11(2):785-92.

  [2]. Wada T, et al. Prevents podocyte apoptosis induced by puromycin aminonucleoside: role of p53 and Bcl-2-related family proteins. J Am Soc Nephrol. 2005 Sep;16(9):2615-25.

  [3]. Gong W, et al. Estrogen-related receptor-α mediates puromycin aminonucleoside-induced mesangial cell apoptosis and inflammatory injury. Am J Physiol Renal Physiol. 2019 May 1;316(5):F906-F913.

  [4]. Xia L, et al. Podocyte-specific expression of organic cation transporter PMAT: implication in puromycin aminonucleosidenephrotoxicity. Am J Physiol Renal Physiol. 2009 Jun;296(6):F1307-13.

  [5]. Kawakami H, et al. Dynamics of absolute amount of nephrin in a single podocyte in puromycin aminonucleoside nephrosis rats calculated by quantitative glomerular proteomics approach with selected reaction monitoring mode. Nephrol Dial Transplant. 2012 Apr;

  [6]. Nosaka K, et al. An adenosine deaminase inhibitor prevents puromycin aminonucleoside nephrotoxicity. Free Radic Biol Med 1997 ;22 (4): 597-605.

Cells are seeded in MEM with 10% FBS on 96-well plates at a density of 5,000 cells/well. After appr 48-h incubation (appr 40-50% confluence), cells are changed to fresh growth medium containing Puromycin aminonucleoside (NSC 3056) at various concentrations. For the protection experiment, cells are incubated in medium containing 250 μM Puromycin aminonucleoside (NSC 3056) with or without the PMAT inhibitor decynium-22 (2 μM). After a total of 72-h incubation in a 95% O₂ incubator at 37°C, cells are washed and the plates. The IC₅₀ values are determined by fitting the cell growth data to the following model using nonlinear regression (WinNonLin version 3.2): S=S_max − [S_max − S₀] × [C_γ/(C_γ + IC_50γ)], where S is the cell survival expressed as percentage of the optical density to untreated control cells, S_max is the maximal cell survival, S₀ is the lowest residual cell survival at the high drug concentration, C is Puromycin aminonucleoside concentration, γ is the Hill coefficient, and IC₅₀ is the Puromycin aminonucleoside concentration leading to half-maximal cell survival. Five to six determinations are carried out within each experiment, and four independent experiments are performed.

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

Male F344 rats at 11 weeks of age are purchased from JaPuromycin aminonucleoside SLC. Normal rats and a Puromycin aminonucleoside nephrosis model are used in the present study. Puromycin aminonucleoside (NSC 3056) nephrosis is induced in rats by a single intravenous injection of Puromycin aminonucleoside at a dose of 8 mg/100 g body weight in saline. Control animals receive an identical volume of saline. Nephrotic rats (n=6 per group) are studied at Days 4 and 7 after the Puromycin aminonucleoside injection.

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Lacalle RA, et al. Cloning of the complete biosynthetic gene cluster for an aminonucleoside antibiotic, puromycin, and its regulated expression in heterologous hosts. EMBO J. 1992 Feb;11(2):785-92.

  [2]. Wada T, et al. Prevents podocyte apoptosis induced by puromycin aminonucleoside: role of p53 and Bcl-2-related family proteins. J Am Soc Nephrol. 2005 Sep;16(9):2615-25.

  [3]. Gong W, et al. Estrogen-related receptor-α mediates puromycin aminonucleoside-induced mesangial cell apoptosis and inflammatory injury. Am J Physiol Renal Physiol. 2019 May 1;316(5):F906-F913.

  [4]. Xia L, et al. Podocyte-specific expression of organic cation transporter PMAT: implication in puromycin aminonucleosidenephrotoxicity. Am J Physiol Renal Physiol. 2009 Jun;296(6):F1307-13.

  [5]. Kawakami H, et al. Dynamics of absolute amount of nephrin in a single podocyte in puromycin aminonucleoside nephrosis rats calculated by quantitative glomerular proteomics approach with selected reaction monitoring mode. Nephrol Dial Transplant. 2012 Apr;

  [6]. Nosaka K, et al. An adenosine deaminase inhibitor prevents puromycin aminonucleoside nephrotoxicity. Free Radic Biol Med 1997 ;22 (4): 597-605.