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Vacuolin-1

Cat No.:V12089 Purity: ≥98%
Vacuolin-1 is a potent cell-penetrable lysosomal exocytosis inhibitor.
Vacuolin-1
Vacuolin-1 Chemical Structure CAS No.: 351986-85-1
Product category: New1
This product is for research use only, not for human use. We do not sell to patients.
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5mg
10mg
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Product Description
Vacuolin-1 is a potent cell-penetrable lysosomal exocytosis inhibitor. Vacuolin-1 blocks lysosomal Ca2+-dependent exocytosis, preventing the release of lysosomal contents without affecting the resealing process. Vacuolin-1 is a potent and specific PIKfyve inhibitor that can suppress late autophagy by impairing lysosomal maturation.
Biological Activity I Assay Protocols (From Reference)
ln Vitro
Ionomycin-induced lysosomal exocytosis is inhibited by vacuolin-1 (1 μM; 1 h pretreatment), but amplified exocytosis is unaffected. It totally suppresses Lamp-1's surface expression and induces lysosomal vacuolation (A) [1]. The inner and limiting membrane properties of endosomes and lysosomes are disrupted by vacuolin-1 (1 μM; 20-180 min). Vacuolin-1 enhances the union of the inner membrane and the limiting membrane, according to ultrastructural studies [1]. β-hexosaminidase release from lysosomes that is reliant on Ca2+ is inhibited by vacuolin-1 (5 or 10 μM; 2 hours). Ionomycin is predicted to release 18–20% of lysosomal β-hexosaminidase in HeLa cells. In contrast to cells not exposed to ionomycin (4%) no more β-hexosaminidase was produced when cells were pretreated with vacuolin-1 [1].
References

[1]. The small chemical vacuolin-1 inhibits Ca(2+)-dependent lysosomal exocytosis but not cell resealing. EMBO Rep. 2004 Sep;5(9):883-8.

[2]. Vacuolin-1 inhibits autophagy by impairing lysosomal maturation via PIKfyve inhibition. FEBS Lett. 2016 Jun;590(11):1576-85.

[3]. Vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating RAB5A. Autophagy.

[4]. Vesicle trafficking plays a novel role in erythroblast enucleation. Blood. 2010 Oct 28;116(17):3331-40.

These protocols are for reference only. InvivoChem does not independently validate these methods.
Physicochemical Properties
Molecular Formula
C26H24N7OI
Molecular Weight
577.41906
Exact Mass
577.108
CAS #
351986-85-1
PubChem CID
9661141
Appearance
White to off-white solid powder
Density
1.5±0.1 g/cm3
Boiling Point
719.7±70.0 °C at 760 mmHg
Flash Point
389.1±35.7 °C
Vapour Pressure
0.0±2.3 mmHg at 25°C
Index of Refraction
1.706
LogP
4.41
Hydrogen Bond Donor Count
1
Hydrogen Bond Acceptor Count
8
Rotatable Bond Count
7
Heavy Atom Count
35
Complexity
634
Defined Atom Stereocenter Count
0
SMILES
C1COCCN1C2=NC(=NC(=N2)N/N=C/C3=CC(=CC=C3)I)N(C4=CC=CC=C4)C5=CC=CC=C5
InChi Key
JMEJTSRAQUFNOP-TURZUDJPSA-N
InChi Code
InChI=1S/C26H24IN7O/c27-21-9-7-8-20(18-21)19-28-32-24-29-25(33-14-16-35-17-15-33)31-26(30-24)34(22-10-3-1-4-11-22)23-12-5-2-6-13-23/h1-13,18-19H,14-17H2,(H,29,30,31,32)/b28-19+
Chemical Name
2-N-[(E)-(3-iodophenyl)methylideneamino]-6-morpholin-4-yl-4-N,4-N-diphenyl-1,3,5-triazine-2,4-diamine
HS Tariff Code
2934.99.9001
Storage

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.
Shipping Condition
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
Solubility Data
Solubility (In Vitro)
DMSO : ~10 mg/mL (~17.32 mM)
Solubility (In Vivo)
Solubility in Formulation 1: 1 mg/mL (1.73 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

Solubility in Formulation 2: ≥ 1 mg/mL (1.73 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

 (Please use freshly prepared in vivo formulations for optimal results.)
Preparing Stock Solutions 1 mg 5 mg 10 mg
1 mM 1.7318 mL 8.6592 mL 17.3184 mL
5 mM 0.3464 mL 1.7318 mL 3.4637 mL
10 mM 0.1732 mL 0.8659 mL 1.7318 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.

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Note: Chemical formula is case sensitive: C12H18N3O4  c12h18n3o4
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In vivo Formulation Calculator (Clear solution)
Step 1: Enter information below (Recommended: An additional animal to make allowance for loss during the experiment)
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Calculation results

Working concentration mg/mL;

Method for preparing DMSO stock solution mg drug pre-dissolved in μL DMSO (stock solution 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 stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O,mix and clarify.

(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
             (2) Be sure to add the solvent(s) in order.

Biological Data
  • Origin of the vacuoles induced by vacuolin-1. BSC-1 cells were labelled with the indicated markers in the absence (left) or following 1 h incubation with 1 μM vacuolin-1 (right). Vacuolin-1 exhibits a vacuolating effect specific for endosomal and lysosomal compartments, manifested by generation of relatively large vacuoles (early endosomes) and small vacuoles (late endosomes and lysosomes). The identity of the resulting vacuoles is maintained, as the markers for each compartment do not intermix. In contrast, the enlargeosomes, Golgi apparatus, ER and nucleus (4,6-diamidino-2-phenylindole staining, in blue) are not affected, and the vacuoles do not contain markers from these organelles. Antibody markers are specific for early endosomes (EEA1), early and late endosomes (CI-M6PR), late endosomes (CD63 and LBPA), lysosomes (Lamp-1), the Golgi apparatus (Gal-Trans-EGFP), ER (PDI) and enlargeosomes (desmoyokin/AHNAK). Scale bar, 10 μM.[1].The small chemical vacuolin-1 inhibits Ca(2+)-dependent lysosomal exocytosis but not cell resealing. EMBO Rep. 2004 Sep;5(9):883-8.
  • Vacuolin-1 blocks ionomycin-induced exocytosis of lysosomes but not of enlargeosomes. The fluorescent images show the effect of vacuolin-1 on the intracellular distribution in BSC-1 cells of the markers Lamp-1, specific for lysosomes (A), (B), and desmoyokin/AHNAK, specific for enlargeosomes (C), (D). The cells were immunostained before or after permeabilization with 0.05% saponin to identify the fusion of lysosomes and enlargeosomes with the plasma membrane and their intracellular location, respectively. In resting cells, lysosomes stain as cytosolic dots slightly larger than those corresponding to enlargeosomes. After treatment with ionomycin (5 μM, 5 min), both markers shift towards the cell periphery and appear at the surface, reflecting the Ca2+-regulated exocytosis of lysosomes (A), (B) and enlargeosomes (C), (D). Pretreatment with 1 μM vacuolin-1 results in vacuolation of lysosomes (A) and complete inhibition of the surface expression of Lamp-1 (D). In contrast, the appearance (C) and the exocytosis of the enlargeosome marker (D) remain normal. Vacuolin-1 alone does not induce surface expression of any marker. Scale bars, 20 μM.[1].The small chemical vacuolin-1 inhibits Ca(2+)-dependent lysosomal exocytosis but not cell resealing. EMBO Rep. 2004 Sep;5(9):883-8.
  • Vacuolin-1 blocks the Ca2+-dependent release of β-hexosaminidase from lysosomes. HeLa cells were incubated for the indicated times in the presence or absence of vacuolin-1 and then treated or not with 5 μM ionomycin for 10 min. Alternatively, the cells were wounded by gently rolling glass beads on top of the cell monolayer. The media were then exchanged, and the amount of β-hexosaminidase released during 10 min was determined. Data, obtained in quadruplicate, are expressed as average ± standard error normalized to the total cellular content of β-hexosaminidase. The same results were obtained with BSC-1 cells (not shown).[1].The small chemical vacuolin-1 inhibits Ca(2+)-dependent lysosomal exocytosis but not cell resealing. EMBO Rep. 2004 Sep;5(9):883-8.
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