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Cyclopamine (11-Deoxojervine) Hedgehog Signaling Pathway Antagonist

Cat.No.S1146

Cyclopamine (11-deoxojervine) is a specific Hedgehog (Hh) signaling pathway antagonist of Smoothened (Smo) with IC50 of 46 nM in TM3Hh12 cells.
Cyclopamine (11-Deoxojervine) Hedgehog/Smoothened antagonist Chemical Structure

Chemical Structure

Molecular Weight: 411.62

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Quality Control

Batch: Purity: 99.99%
99.99

Cell Culture, Treatment & Working Concentration

Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID
OS-RC-2 Growth Inhibition Assay IC50=5.8666 μM SANGER
DOHH-2 Growth Inhibition Assay IC50=9.35689 μM SANGER
no-10 Growth Inhibition Assay IC50=9.9039 μM SANGER
LS-513 Growth Inhibition Assay IC50=11.3547 μM SANGER
ALL-PO Growth Inhibition Assay IC50=11.7734 μM SANGER
8-MG-BA Growth Inhibition Assay IC50=13.1123 μM SANGER
RPMI-8402 Growth Inhibition Assay IC50=15.8537 μM SANGER
EoL-1-cell Growth Inhibition Assay IC50=18.5948 μM SANGER
NALM-6 Growth Inhibition Assay IC50=19.0167 μM SANGER
DEL Growth Inhibition Assay IC50=20.1471 μM SANGER
SR Growth Inhibition Assay IC50=23.6715 μM SANGER
697 Growth Inhibition Assay IC50=26.6155 μM SANGER
COLO-829 Growth Inhibition Assay IC50=26.8483 μM SANGER
EVSA-T Growth Inhibition Assay IC50=27.5561 μM SANGER
ATN-1 Growth Inhibition Assay IC50=31.2329 μM SANGER
L-363 Growth Inhibition Assay IC50=31.7461 μM SANGER
LAMA-84 Growth Inhibition Assay IC50=32.5211 μM SANGER
NOS-1 Growth Inhibition Assay IC50=34.2956 μM SANGER
BB30-HNC Growth Inhibition Assay IC50=34.3306 μM SANGER
BC-1 Growth Inhibition Assay IC50=37.9746 μM SANGER
IST-SL2 Growth Inhibition Assay IC50=38.224 μM SANGER
D-392MG Growth Inhibition Assay IC50=40.2215 μM SANGER
no-11 Growth Inhibition Assay IC50=40.5521 μM SANGER
LC4-1 Growth Inhibition Assay IC50=40.8716 μM SANGER
A388 Growth Inhibition Assay IC50=42.5848 μM SANGER
NTERA-S-cl-D1 Growth Inhibition Assay IC50=42.7074 μM SANGER
CESS Growth Inhibition Assay IC50=44.2232 μM SANGER
RS4-11 Growth Inhibition Assay IC50=49.0938 μM SANGER
MS-1 Growth Inhibition Assay IC50=50.9351 μM SANGER
CTV-1 Growth Inhibition Assay IC50=51.074 μM SANGER
D-502MG Growth Inhibition Assay IC50=51.6271 μM SANGER
ML-2 Growth Inhibition Assay IC50=52.9195 μM SANGER
SK-NEP-1 Growth Inhibition Assay IC50=53.3923 μM SANGER
LOXIMVI Growth Inhibition Assay IC50=53.5884 μM SANGER
DJM-1 Growth Inhibition Assay IC50=56.3391 μM SANGER
GI-1 Growth Inhibition Assay IC50=56.6149 μM SANGER
IST-MES1 Growth Inhibition Assay IC50=60.5493 μM SANGER
MV-4-11 Growth Inhibition Assay IC50=60.6538 μM SANGER
OVCAR-4 Growth Inhibition Assay IC50=63.5657 μM SANGER
KE-37 Growth Inhibition Assay IC50=66.2668 μM SANGER
D-542MG Growth Inhibition Assay IC50=68.4135 μM SANGER
MHH-PREB-1 Growth Inhibition Assay IC50=72.8441 μM SANGER
MRK-nu-1 Growth Inhibition Assay IC50=73.4705 μM SANGER
D-247MG Growth Inhibition Assay IC50=73.5442 μM SANGER
OCI-AML2 Growth Inhibition Assay IC50=76.9369 μM SANGER
LP-1 Growth Inhibition Assay IC50=82.8731 μM SANGER
HCC1599 Growth Inhibition Assay IC50=84.2837 μM SANGER
KARPAS-45 Growth Inhibition Assay IC50=84.6992 μM SANGER
BE-13 Growth Inhibition Assay IC50=99.0477 μM SANGER
GCIY Growth Inhibition Assay IC50=99.0954 μM SANGER
BV-173 Growth Inhibition Assay IC50=100.325 μM SANGER
LB2518-MEL Growth Inhibition Assay IC50=100.789 μM SANGER
KS-1 Growth Inhibition Assay IC50=101.639 μM SANGER
MOLT-16 Growth Inhibition Assay IC50=104.986 μM SANGER
NCI-H1770 Growth Inhibition Assay IC50=108.784 μM SANGER
NCI-H82 Growth Inhibition Assay IC50=110.976 μM SANGER
NCCIT Growth Inhibition Assay IC50=112.529 μM SANGER
KALS-1 Growth Inhibition Assay IC50=115.941 μM SANGER
LB2241-RCC Growth Inhibition Assay IC50=116.679 μM SANGER
HH Growth Inhibition Assay IC50=117.395 μM SANGER
HD-MY-Z Growth Inhibition Assay IC50=118.488 μM SANGER
EB-3 Growth Inhibition Assay IC50=123.094 μM SANGER
BL-70 Growth Inhibition Assay IC50=123.127 μM SANGER
K-562 Growth Inhibition Assay IC50=126.245 μM SANGER
HT-144 Growth Inhibition Assay IC50=133.164 μM SANGER
PF-382 Growth Inhibition Assay IC50=134.361 μM SANGER
RPMI-8226 Growth Inhibition Assay IC50=135.045 μM SANGER
NCI-H1355 Growth Inhibition Assay IC50=135.587 μM SANGER
LXF-289 Growth Inhibition Assay IC50=139.781 μM SANGER
NCI-H69 Growth Inhibition Assay IC50=142.932 μM SANGER
SK-MEL-1 Growth Inhibition Assay IC50=147.13 μM SANGER
KARPAS-299 Growth Inhibition Assay IC50=149.12 μM SANGER
GB-1 Growth Inhibition Assay IC50=149.322 μM SANGER
CMK Growth Inhibition Assay IC50=149.515 μM SANGER
MPP-89 Growth Inhibition Assay IC50=156.035 μM SANGER
KU812 Growth Inhibition Assay IC50=161.902 μM SANGER
REH Growth Inhibition Assay IC50=162.125 μM SANGER
NEC8 Growth Inhibition Assay IC50=165.026 μM SANGER
KP-N-YS Growth Inhibition Assay IC50=168.395 μM SANGER
Ramos-2G6-4C10 Growth Inhibition Assay IC50=169.915 μM SANGER
Becker Growth Inhibition Assay IC50=174.18 μM SANGER
LB647-SCLC Growth Inhibition Assay IC50=175.845 μM SANGER
LU-139 Growth Inhibition Assay IC50=178.019 μM SANGER
QIMR-WIL Growth Inhibition Assay IC50=179.646 μM SANGER
NCI-H1395 Growth Inhibition Assay IC50=179.996 μM SANGER
NOMO-1 Growth Inhibition Assay IC50=182.85 μM SANGER
GI-ME-N Growth Inhibition Assay IC50=187.969 μM SANGER
KMS-12-PE Growth Inhibition Assay IC50=189.273 μM SANGER
Daudi Growth Inhibition Assay IC50=191.128 μM SANGER
LB996-RCC Growth Inhibition Assay IC50=191.699 μM SANGER
NCI-H2107 Growth Inhibition Assay IC50=193.739 μM SANGER
SK-PN-DW Growth Inhibition Assay IC50=194.719 μM SANGER
MC-CAR Growth Inhibition Assay IC50=202.253 μM SANGER
SNB75 Growth Inhibition Assay IC50=221.94 μM SANGER
ES4 Growth Inhibition Assay IC50=223.783 μM SANGER
KARPAS-422 Growth Inhibition Assay IC50=228.352 μM SANGER
NCI-H1648 Growth Inhibition Assay IC50=229.489 μM SANGER
ES6 Growth Inhibition Assay IC50=239.43 μM SANGER
KNS-81-FD Growth Inhibition Assay IC50=241.197 μM SANGER
JAR Growth Inhibition Assay IC50=256.225 μM SANGER
NB1 Growth Inhibition Assay IC50=260.516 μM SANGER
D-336MG Growth Inhibition Assay IC50=260.698 μM SANGER
BC-3 Growth Inhibition Assay IC50=265.178 μM SANGER
HCC2218 Growth Inhibition Assay IC50=266.415 μM SANGER
TE-9 Growth Inhibition Assay IC50=266.627 μM SANGER
LB1047-RCC Growth Inhibition Assay IC50=266.753 μM SANGER
CTB-1 Growth Inhibition Assay IC50=269.973 μM SANGER
NB7 Growth Inhibition Assay IC50=271 μM SANGER
ST486 Growth Inhibition Assay IC50=277.412 μM SANGER
HCC1187 Growth Inhibition Assay IC50=282.811 μM SANGER
NCI-SNU-16 Growth Inhibition Assay IC50=284.248 μM SANGER
COR-L279 Growth Inhibition Assay IC50=291.584 μM SANGER
ES8 Growth Inhibition Assay IC50=294.182 μM SANGER
U-698-M Growth Inhibition Assay IC50=298.243 μM SANGER
HEL Growth Inhibition Assay IC50=309.149 μM SANGER
KINGS-1 Growth Inhibition Assay IC50=310.674 μM SANGER
KY821 Growth Inhibition Assay IC50=336.595 μM SANGER
MZ1-PC Growth Inhibition Assay IC50=345.618 μM SANGER
LS-411N Growth Inhibition Assay IC50=354.66 μM SANGER
SIG-M5 Growth Inhibition Assay IC50=359.782 μM SANGER
HT Growth Inhibition Assay IC50=367.711 μM SANGER
HC-1 Growth Inhibition Assay IC50=367.787 μM SANGER
NCI-H1694 Growth Inhibition Assay IC50=372.934 μM SANGER
BB65-RCC Growth Inhibition Assay IC50=376.245 μM SANGER
HAL-01 Growth Inhibition Assay IC50=379.838 μM SANGER
ARH-77 Growth Inhibition Assay IC50=394.008 μM SANGER
MZ7-mel Growth Inhibition Assay IC50=397.233 μM SANGER
SIMA Growth Inhibition Assay IC50=403.933 μM SANGER
DG-75 Growth Inhibition Assay IC50=415.698 μM SANGER
HUTU-80 Growth Inhibition Assay IC50=419.185 μM SANGER
KNS-42 Growth Inhibition Assay IC50=425.815 μM SANGER
SH-4 Growth Inhibition Assay IC50=427.565 μM SANGER
L-540 Growth Inhibition Assay IC50=431.031 μM SANGER
NB10 Growth Inhibition Assay IC50=441.234 μM SANGER
ES1 Growth Inhibition Assay IC50=452.753 μM SANGER
KMOE-2 Growth Inhibition Assay IC50=456.711 μM SANGER
MC116 Growth Inhibition Assay IC50=458.116 μM SANGER
RCC10RGB Growth Inhibition Assay IC50=460.005 μM SANGER
RL95-2 Growth Inhibition Assay IC50=460.237 μM SANGER
Raji Growth Inhibition Assay IC50=468.143 μM SANGER
CAS-1 Growth Inhibition Assay IC50=472.073 μM SANGER
Calu-6 Growth Inhibition Assay IC50=475.265 μM SANGER
KG-1 Growth Inhibition Assay IC50=478.44 μM SANGER
LB771-HNC Growth Inhibition Assay IC50=482.232 μM SANGER
ACN Growth Inhibition Assay IC50=493.599 μM SANGER
KM12 Growth Inhibition Assay IC50=496.589 μM SANGER
U2OS Function assay Binding affinity to wild type Smo expressed in U2OS cells by scintillation counting, Kd = 0.0124 μM. 23063522
U2OS Function assay 2 hrs Displacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation counting, Ki = 0.0127 μM. 23063522
TM3 Function assay 48 hrs Inhibition of Hedgehog signaling pathway in mouse TM3 cells bearing pTA-8xGli-Luc reporter construct assessed as transcriptional modulation of Gli after 48 hrs by luciferase assay, IC50 = 0.046 μM. 19091559
HCC827 Function assay Displacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation counting, Ki = 0.051 μM. 28787156
HEK293 Function assay 2 hrs Displacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopy, IC50 = 0.064 μM. 22268551
Shh Light2 Function assay 30 hrs Inhibition of hedgehog signaling pathway in mouse Shh Light2 cells assessed as inhibition of sonic hedgehog-induced GLI1-mediated transcriptional activity measured after 30 hrs by dual luciferase reporter gene assay, IC50 = 0.0741 μM. 27567371
U2OS Function assay Binding affinity to Smo D473H mutant expressed in U2OS cells by scintillation counting, Kd = 0.116 μM. 23063522
DaOY Function assay 48 hrs Inhibition of Hedgehog signaling in human DaOY cells assessed as downregulation of Gli1 mRNA expression after 48 hrs by RT-PCR analysis, IC50 = 0.16 μM. 24900716
U2OS Function assay 2 hrs Displacement of [3H]cyclopamine from Smo D473H mutant expressed in U2OS cells after 2 hrs by scintillation counting, Ki = 0.232 μM. 23063522
CHO Function assay Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay, IC50 = 0.28 μM. 19091559
Shh Light2 Function assay 40 hrs Inhibition of SHH pathway in mouse Shh Light2 cells after 40 hrs by Gli-dependent luciferase reporter gene assay, IC50 = 0.3 μM. 21592788
Shh-light2 Function assay Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay, IC50 = 0.3 μM. 22268551
M210B4 Function assay 24 hrs Inhibition of Hedgehog signaling in mouse M210B4 cells assessed as downregulation of Ptch mRNA expression after 24 hrs by RT-PCR analysis, IC50 = 0.43 μM. 24900716
Shh-Light 2 Function assay 2 days Antagonist activity at Smo in mouse Shh-Light 2 cells assessed as inhibition of Shh-induced Gli1-reporter activity after 2 days by dual-luciferase reporter gene method, IC50 = 0.484 μM. 23063522
Shh Light2 Function assay Inhibition of SHH in mouse Shh Light2 cells by GLI-responsive firefly luciferase reporter gene assay, EC50 = 0.5 μM. 19309080
C3H10T1/2 Function assay Inhibition of N-terminal SHH activated pathway in mouse C3H10T1/2 cells assessed as SAG-induced cell differentiation by alkaline phosphatase assay, IC50 = 0.6 μM. 21592788
C3H10T1/2 Function assay 6 hrs Inhibition of SAG-induced differentiation of mouse mesenchymal pluripotent C3H10T1/2 cells to alkaline phosphatase positive oeseoblasts after 6 hrs, IC50 = 0.62 μM. 22268551
ASZ001 Function assay 48 hrs Inhibition of Hedgehog signaling in mouse ASZ001 cells assessed as downregulation of Gli1 mRNA expression after 48 hrs by RT-PCR analysis, IC50 = 0.66 μM. 24900716
M210B4 Function assay 24 hrs Inhibition of Hedgehog signaling in mouse M210B4 cells assessed as downregulation of Gli1 mRNA expression after 24 hrs by RT-PCR analysis, IC50 = 0.8 μM. 24900716
medulloblastoma cells Antiproliferative assay Antiproliferative activity against mouse medulloblastoma cells harboring heterozygous ptch1 gene by MTT assay, EC50 = 1 μM. 17417631
CHO Function assay Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay, IC50 = 1.2 μM. 19091559
Shh-Light2 Function assay 48 hrs Inhibition of SHH in mouse Shh-Light2 cells after 48 hrs by Gli1 reporter gene assay in presence of SAG, IC50 = 1.312 μM. 19541490
MEF Function assay Inhibition of Smo in mouse Ptch-deficient MEF cells assessed as inhibition of Shh-induced Gli1 transcriptional activity, IC50 = 1.5 μM. 23074541
MEF Function assay 30 hrs Inhibition of Smo in mouse Ptch-deficient MEF cells assessed as inhibition of Shh-induced Gli-responsive betagalactosidase activity after 30 hrs by BetaGLo assay, IC50 = 1.9 μM. 23074541
neural precursor cells Antiproliferative assay Antiproliferative activity against mouse neural precursor cells by colony formation assay, EC50 = 13.44 μM. 17417631
U87 Cytotoxicity assay Cytotoxicity against human U87 cells assessed as viability in presence of beta glucuronidase, IC50 = 15.5 μM. 20116904
U87MG Antiproliferative assay 72 hrs Antiproliferative activity against human U87MG cells after 72 hrs by MTS assay, IC50 = 22.5 μM. 22226657
A549 Anticancer assay Anticancer activity against human A549 cells by MTS assay, IC50 = 49 μM. 18221872
DU145 Growth inhibition assay 5 uM 96 hrs Growth inhibition of human DU145 cells at 5 uM after 96 hrs 18249125
DU145 Growth inhibition assay 10 uM 96 hrs Growth inhibition of human DU145 cells at 10 uM after 96 hrs 18249125
HEK293 Function assay Inhibition of beta galactosidase in HEK293 cells 17494766
22Rv Function assay Reduction of expression of PTCH mRNA in human 22Rv cells 17494766
PANC1 Function assay 0.2 uM 24 hrs Inhibition of Hh/GLI1-mediated PTCH mRNA expression in human PANC1 cells at 0.2 uM after 24 hrs by RT-PCR 20450170
PANC1 Function assay 0.4 uM 24 hrs Inhibition of Hh/GLI1-mediated PTCH mRNA expression in human PANC1 cells at 0.4 uM after 24 hrs by RT-PCR 20450170
Shh Light2 Function assay 6.25 uM 30 hrs Inhibition of N-palmitoylated Shh in mouse Shh Light2 cells at 6.25 uM after 30 hrs by firefly luciferase reporter gene assay 19151731
U87MG Function assay 10 uM 4 hrs Inhibition of Hedgehog signaling pathway in human U87MG cells assessed as down regulation of Gli1 at 10 uM after 4 hrs by RT-PCR analysis 22226657
HEK293 Function assay 5 uM 10 hrs Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells at 5 uM measured after 10 hrs by DAPI staining based fluorescence microscopic assay 27736063
HEK293 Function assay 5 uM 10 hrs Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells at 5 uM measured after 10 hrs by FACS analysis 27736063
C3H10T1/2 Function assay 55.5 to 4500 nM Competitive inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of SAG-induced Gli1 transcriptional activity at 55.5 to 4500 nM by qPCR analysis 23074541
U2OS Function assay 5 uM 6 hrs Antagonist activity at chimeric Smo 633 mutant expressed in U2OS cells coexpressing beta arrestin2-GFP assessed as inhibition of intracellular beta arrestin2-GFP aggregate formation at 5 uM after 6 hrs by confocal microscopy 23063522
Click to View More Cell Line Experimental Data

Solubility

In vitro
Batch:

Ethanol : 28 mg/mL

DMSO : Insoluble
(Moisture-contaminated DMSO may reduce solubility. Use fresh, anhydrous DMSO.)

Water : Insoluble

Molarity Calculator

Mass Concentration Volume Molecular Weight
Dilution Calculator Molecular Weight Calculator

In vivo
Batch:

In vivo Formulation Calculator (Clear solution)

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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.
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Chemical Information, Storage & Stability

Molecular Weight 411.62 Formula

C27H41NO2

 Storage (From the date of receipt)
CAS No. 4449-51-8 Download SDF Storage of Stock Solutions

Synonyms 11-deoxojervine Smiles CC1CC2C(C(C3(O2)CCC4C5CC=C6CC(CCC6(C5CC4=C3C)C)O)C)NC1

Mechanism of Action

Targets/IC50/Ki
Smoothened
(TM3Hh12 cells)
46 nM
In vitro
Cyclopamine inhibits the Hedgehog signaling pathway with an IC50 of 46 nM, and blocks the activity of human Smo receptor expressed in CHO-K1 cells in [3H]Hh-Ag binding assay with an IC50 of 280 nM. This compound significantly inhibits Hedgehog pathway activity in a dose-dependent manner in gut-derived tumor cell lines expressing Patched (PTCH) mRNA, and induces growth inhibition of those tumor cell lines by 75-95% at the concentration of 3 μM, but ineffective towards the colon tumor cells without PTCH mRNA expression, suggesting the effects of this chemical treatment are Hedgehog pathway related rather than generally cytotoxic. By blocking Hedgehog signaling through direct interaction with Smo, it (10 μM) inhibits the proliferation of SMOhigh Cyclopamine-responsive cell lines L3.6sl and Panc 05.04 by 75-80%, and increases the apoptosis by 2.5- to 3.5-fold, without affecting the BxPC3-SMOlow cell line. This treatment significantly decreases of Snail mRNA and increasea E-cadherin transcripts in the E3LZ10.7 cell line. Independent of inhibition of cell growth, it significantly inhibits the invasive phenotype of Hedgehog-dependent L3.6pl cells, causing a >500-fold reduction in the number of transmigrating cells, but not that of the Hedgehog-independent cell line Panc-1.
Kinase Assay
Hedgehog cell assay
This assay measures the end stage of the Hh signaling pathway, that is, the transcriptional modulation of Gli, using Luciferase as readout (Gli-Luc assay). Cyclopamine is prepared for assay by serial dilution in DMSO and then added to empty assay plates. TM3Hh12 cells (TM3 cells containing Hh-responsive reporter gene construct pTA-8xGli-Luc) are resuspended in F12 Ham's/DMEM (1:1) containing 5% FBS and 15 mM Hepes pH 7.3, added to assay plates and incubated with this compound for approximately 30 minutes at 37 °C in 5% CO2. 1 nM Hh-Ag 1.5 is then added to assay plates and incubated at 37 °C in the presence of 5% CO2. After 48 hours, either Bright-Glo or MTS reagent is added to the assay plates and luminescence or absorbance at 492 nm is determined. IC50 value, defined as the inflection point of the logistic curve, is determined by non-linear regression of the Gli-driven luciferase luminescence or absorbance signal from MTS assay vs log10 (concentration) of this chemical using the R statistical software pack
In vivo
Administration of Cyclopamine at dose of 50 mg/kg/day for 22 days eradicates the HUCCT1 xenografts in mice with no obvious adverse effects. This compound treatment at dose of 1.2 mg for 7 days induces significant apoptosis of tumor cells and decreases the tumor mass by 50-60% in Panc 05.04- and L3.6sl-derived tumors, respectively, but not in the BxPC3-SMOlow tumors. [3] Administration of this chemical alone profoundly inhibits tumor metastases in xenografts of E3LZ10.7 and L3.6pl, and completely abrogates metastases when in combination of gemcitabine.
References
  • [4] https://pubmed.ncbi.nlm.nih.gov/17332349/

Applications

Methods Biomarkers Images PMID
Western blot Snail / E-cadherin / Slug / Vimentin Gli1 / TGF-β1 / CXCR4 NF-κB / Cyclin D1 / MMP2 / MMP9
S1146-WB3
26859575
Immunofluorescence PCNA / β-catenin Vimentin
S1146-IF2
28747625

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Frequently Asked Questions

Question 1:
How to reconstitute it for in vivo use in mice?

Answer:
One paper dissolved this compound in DMSO, and diluted it in saline: Berman DM, et al. Nature, 2003, 425(6960), 846-851. Alternatively, you can try this vehicle: 10% DMSO+30% PEG 300+5% Tween 80+ddH2O for P.O. When preparing the solution, please dissolve it in DMSO clearly first. Then add PEG300 and Tween, after they mixed well, dilute with water.

Signaling Pathway Map