Name: NSC 74859 (S3I-201)
Brand: Selleck Chemicals
SKU: S1155
Availability: InStock
Rating: 5 (242 reviews)

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Quality Control

Batch: Purity: 99.28%

99.28

Related Targets	EGFR JAK Pim
Other STAT Inhibitors	Napabucasin (BBI608) Stattic Cryptotanshinone (Tanshinone C) SH-4-54 C188-9 (TTI-101) BP-1-102 HO-3867 Nifuroxazide AS1517499 Homoharringtonine (HHT)

Cell Culture, Treatment & Working Concentration

Cell Lines	Assay Type	Concentration	Incubation Time	Formulation	Activity Description	PMID
U87	Growth Inhibition Assay		72 h		IC50=55.1 μM	20072652
U373	Growth Inhibition Assay		72 h		IC50=52.5 μM	20072652
HPAC	Growth Inhibition Assay		72 h		IC50＞100 μM	20072652
PANC-1	Growth Inhibition Assay		72 h		IC50＞100 μM	20072652
SK-BR-3	Growth Inhibition Assay		72 h		IC50＞100 μM	20072652
U-373 MG	Cytotoxicity Assay	3/10 μM	24 h		reduces FN-γ-induced cell neurotoxicity	20888416
MDA-MB-231	Growth Inhibition Assay		72 h		IC50＞100 μM	20072652
HUVEC	Function Assay	0.5-20 μM	24 h	DMSO	suppresses the hypoxia-induced accumulation of HIF-1α	21523559
Huh7	Growth Inhibition Assay	100 nM	48 h	DMSO	inhibits the IL-6 stimulation promoted cell proliferation	23364389
PLC/PRF/5	Growth Inhibition Assay	100 nM	48 h	DMSO	inhibits the IL-6 stimulation promoted cell proliferation	23364389
H460	Function Assay	50/100 μM	48 h		inhibits the Stat3C increased miR-92a expression	23820254
H1299	Function Assay	50/100 μM	48 h		suppresses miR-92a expression dose-dependently	23820254
T-cell	Growth Inhibition Assay				IC50=50 μM	24068731
U373	Growth Inhibition Assay	125 μM	24 h	DMSO	disrupts STAT3 signaling and proliferation	24070820
HUT-102	Apoptosis Assay	75-300 μM	24/48 h		suppresses cell proliferation in a dose-dependent manner and induces cell apoptosis	24090995
MT-2	Apoptosis Assay	75-300 μM	24/48 h		suppresses cell proliferation in a dose-dependent manner and induces cell apoptosis	24090995
H460	Apoptosis Assay	100 nM	24 h		enhances cell death co-treated with LY294002	24472538
A459	Apoptosis Assay	100 nM	24 h		induces cell apoptosis co-treated with BEZ235	24472538
H460	Apoptosis Assay	100 nM	24 h		induces cell apoptosis co-treated with BEZ235	24472538
GC	Growth Inhibition Assay	50-125 μM	72 h		attenuates the cell growth in a dose-dependent manner	25774503
GH3	Growth Inhibition Assay	50-125 μM	72 h		attenuates the cell growth in a dose-dependent manner	25774503
BT474R	Function Assay	50 μM	10-60 d		inhibits STAT3 activity	25327561
NCI-N87R	Function Assay	50 μM	10-60 d		inhibits STAT3 activity	25327561
MDA-MB-468	Function assay	100 uM	24 hrs		Inhibition of Stat3 activation in human MDA-MB-468 cells at 100 uM after 24 hrs	17463090
MDA-MB-435	Function assay	100 uM	24 hrs		Inhibition of Stat3 activation in human MDA-MB-435 cells at 100 uM after 24 hrs	17463090
MDA-MB-231	Function assay	100 uM	24 hrs		Inhibition of Stat3 activation in human MDA-MB-231 cells at 100 uM after 24 hrs	17463090
NIH3T3	Function assay	100 uM	24 hrs		Reduction of pTyr-705 Stat3 level in v-Src expressing mouse NIH3T3 cells at 100 uM after 24 hrs	17463090
NIH3T3	Growth inhibition assay	100 uM	4 days		Growth inhibition of mouse NIH3T3 cells expressing v-Src at 100 uM after 4 days by trypan blue exclusion assay	17463090
MDA-MB-435	Growth inhibition assay	100 uM	4 days		Growth inhibition of human MDA-MB-435 cells expressing v-Src at 100 uM after 4 days by trypan blue exclusion assay	17463090
MDA-MB-231	Growth inhibition assay	100 uM	4 days		Growth inhibition of human MDA-MB-231 cells expressing v-Src at 100 uM after 4 days by trypan blue exclusion assay	17463090
MDA-MB-468	Growth inhibition assay	100 uM	4 days		Growth inhibition of human MDA-MB-468 cells expressing v-Src at 100 uM after 4 days by trypan blue exclusion assay	17463090
NIH3T3	Growth inhibition assay	100 uM			Growth inhibition of mouse NIH3T3 cells expressing v-Ras at 100 uM for every 3 days by soft-agar colony-formation assay	17463090
MDA-MB-435	Apoptosis assay	30 to 100 uM	48 hrs		Induction of apoptosis in human MDA-MB-435 cells expressing active Stat3 at 30 to 100 uM after 48 hrs	17463090
MDA-MB-231	Apoptosis assay	100 uM	24 hrs		Reduction of apoptosis in Stat3 transfected human MDA-MB-231 cells at 100 uM after 24 hrs	17463090
MDA-MB-231	Function assay	100 uM	48 hrs		Reduction of cyclin D1 gene expression in human MDA-MB-231 cells at 100 uM after 48 hrs	17463090
MDA-MB-231	Apoptosis assay	100 uM	24 hrs		Induction of apoptosis in Stat3 SH2 domain transfected human MDA-MB-231 cells at 100 uM after 24 hrs	17463090
MDA-MB-231	Apoptosis assay	100 uM	24 hrs		Induction of apoptosis in Stat3C transfected human MDA-MB-231 cells at 100 uM after 24 hrs	17463090
NIH3T3	Function assay	100 uM	48 hrs		Reduction of cyclin D1 gene expression in v-Src transfected mouse NIH3T3 cells at 100 uM after 48 hrs	17463090
NIH3T3	Function assay	100 uM	48 hrs		Reduction of Bcl-xL gene expression in v-Src transfected mouse NIH3T3 cells at 100 uM after 48 hrs	17463090
NIH3T3	Function assay	100 uM	48 hrs		Reduction of survivin gene expression in v-Src transfected mouse NIH3T3 cells at 100 uM after 48 hrs	17463090
MDA-MB-231	Function assay	100 uM	48 hrs		Reduction of Bcl-xL gene expression in human MDA-MB-231 cells at 100 uM after 48 hrs	17463090
MDA-MB-231	Function assay	100 uM	48 hrs		Reduction of survivin gene expression in human MDA-MB-231 cells at 100 uM after 48 hrs	17463090
MDA-MB-231	Antitumor assay	5 mg/kg	2 weeks		Antitumor activity against human MDA-MB-231 cells expressing active Stat3 xenografted in mouse at 5 mg/kg, iv for every 3 days for 2 weeks	17463090
NIH3T3	Growth inhibition assay	100 uM			Growth inhibition of mouse NIH3T3 cells expressing v-Src at 100 uM for every 3 days by soft-agar colony-formation assay	17463090
A673	qHTS assay				qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for A673 cells	29435139
SK-N-MC	qHTS assay				qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for SK-N-MC cells	29435139
NB1643	qHTS assay				qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for NB1643 cells	29435139
LAN-5	qHTS assay				qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for LAN-5 cells	29435139
Click to View More Cell Line Experimental Data

Solubility

In vitro
Batch:

DMSO : 73 mg/mL (199.8 mM)
(Moisture-contaminated DMSO may reduce solubility. Use fresh, anhydrous DMSO.)

Water : Insoluble

Ethanol : Insoluble

Molarity Calculator

Mass	Concentration	Volume	Molecular Weight
Mass value Mass unit	Concentration value Concentration unit	Volume value Volume unit	Molecular weight (g/mol)

Dilution Calculator Molecular Weight Calculator

In vivo batch selection In vivo Batch:
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.
Clear solution	5%DMSO 1 Corn oil Validated by Selleck labs. Should you need adjustments to this formulation, contact our sales team for custom testing.	3.000mg/ml (8.21mM)	Taking the 1 mL working solution as an example, add 50 μL of 60 mg/ml clear DMSO stock solution to 950 μL of corn oil and mix evenly. The mixed solution should be used immediately for optimal results.
Clear solution	2% DMSO 1 40% PEG 300 2 5%Tween80 3 53%ddH2O Validated by Selleck labs. Should you need adjustments to this formulation, contact our sales team for custom testing.	1.460mg/ml (4.00mM)	Taking the 1 mL working solution as an example, add 20 μL of 73 mg/ml clarified DMSO stock solution to 400 μL of PEG300, mix evenly to clarify it; add 50 μL of Tween80 to the above system, mix evenly to clarify; then continue to add 530 μL of ddH2O to adjust the volume to 1 mL. The mixed solution should be used immediately for optimal results.

In vivo Formulation Calculator (Clear solution)

Step 1: Enter information below (Recommended: An additional animal making an allowance for loss during the experiment)

Dosage: mg/kg

Average weight of animals: g

Dosing volume per animal: μL

Number of animals:

Step 2: Enter the in vivo formulation (This is only the calculator, not formulation. Please contact us first if there is no in vivo formulation at the solubility Section.)

% DMSO

%

% Tween 80

% ddH₂O

% DMSO

+

%

Calculation results:

Working concentration: mg/ml;

Method for preparing DMSO master liquid: mg drug pre-dissolved in μL DMSO ( Master liquid concentration mg/mL, Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug. )

Method for preparing in vivo formulation: Take μL DMSO master liquid, next addμL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH₂O, mix and clarify.

Method for preparing in vivo formulation: Take μL DMSO master liquid, next add μL Corn oil, mix and clarify.

Note: 1. Please make sure the liquid is clear before adding the next solvent.
2. Be sure to add the solvent(s) in order. You must ensure that the solution obtained, in the previous addition, is a clear solution before proceeding to add the next solvent. Physical methods such
as vortex, ultrasound or hot water bath can be used to aid dissolving.

Chemical Information, Storage & Stability

Molecular Weight	365.36	Formula	C₁₆H₁₅NO₇S	Storage (From the date of receipt)	3 years-20°Cpowder
CAS No.	501919-59-1	Download SDF		Storage of Stock Solutions	1 year-80°Cin solvent 1 month-20°Cin solvent

Mechanism of Action

Features	A chemical probe inhibitor of Stat3 activity.
Targets/IC₅₀/Ki	STAT3 (Cell-free assay) 86 μM
In vitro	NSC 74859 (S3I-201) inhibits growth and induces apoptosis preferentially in tumor cells that contain persistently activated Stat3 by inhibiting Stat3·Stat3 complex formation and Stat3 DNA-binding and transcriptional activities. Moreover, it also inhibits the expression of the Stat3-regulated genes encoding cyclin D1, Bcl-xL, and survivin. This compound inhibits breast carcinoma MDA-MB-435, MDA-MB-453 and MDA-MB-231 cell lines with IC50 of 100 μM. In addition, the cells with impaired TGF-β signaling are four times as sensitive to S3I-201. A recent study shows that it potentiates the antiproliferative effect in HepG2 and Huh-7 cells via the STAT3 signalling pathway.
Kinase Assay	In vitro Stat3 DNA-binding assay and EMSA analysis
Kinase Assay	Briefly, 100 mL of biotinyl-e-Ac-EPQpYEEIEL-OH (in 50 mM Tris/150 mM NaCl, pH 7.5) is added to each well of streptavidin-coated 96-well microtiter plates and incubated with shaking at 4 °C overnight. Then plates are rinsed with PBS/Tween 20 and then two times with 200 mL of BSA-T-PBS (0.2% BSA/0.1% Tween 20/PBS). Then 50 mL of Lck-SH2-GST fusion protein (6.4 ng/ml in BSA-T-PBS) is added to each well of the 96-well plate in the presence and absence of 50 mL of NSC 74859 (S3I-201) (for 30 and 100 mM final concentrations), and the plate is shaken at room temperature for 4 hours. After solutions are removed, each well is rinsed four times with BSA-T-PBS (200 mL), and 100 mL of polyclonal rabbit anti-GST antibody (100 ng/mL in BSA-T-PBS) is added to each well and incubated at 4 °C overnight. After washing with BSA-T-PBS, 100 mL of 200 ng/mL BSA-T-PBS horseradish peroxidase-conjugated mouse anti-rabbit antibody is added to each well and incubated for 45 minutes at room temperature. After four washing steps with BSA-T-PBS and three washing steps with PBS-T, 100 mL of peroxidase substrate is added to each well and incubated for 5-15 minutes. The peroxidase reaction is stopped by adding 100 mL of 1 M sulfuric acid solution, and absorbance is read at 450 nm with an ELISA plate rea
In vivo	NSC 74859 (S3I-201) (5 mg/kg, i.v. every 2 or every 3 days) shows the antitumor efficacy in mouse models with human breast tumor xenografts that harbor constitutively active Stat3. This compound treatment reduces Varicella-zoster virus (VZV) replication on the basis of the bioluminescence signal and the number of positive skin xenografts compared with DMSO-treated mice by inhibiting STAT3 phosphorylation.
References	[1] https://pubmed.ncbi.nlm.nih.gov/17463090/ [2] https://pubmed.ncbi.nlm.nih.gov/19137011/ [3] https://pubmed.ncbi.nlm.nih.gov/22098470/ [4] https://pubmed.ncbi.nlm.nih.gov/22190485/

Applications

Methods	Biomarkers	PMID
Western blot	STAT3 / p-STAT3 / Cyclin D1 / Bcl-2 / Nanog / OCT4 / ALDH1 / CD44 PD-L1	26556875
Immunofluorescence	p-STAT3 Oct4 / Twist	26556875
Growth inhibition assay	Cell viability	26813676