CME/clathrin-mediated endocytosis

Pitstop 2 (20–40 μM; 30 min) prevents J774A.1 macrophages from endocytosing transferrin while maintaining cell viability[1]. Pitstop 2 (20–40 μM; 30 min) has no effect on J774A.1 macrophages' ability to internalize cholera toxin B[1]. Pitstop 2 (0.001-100 μM; 6 h) inhibits the spindle of HeLa cells, preventing them from progressing through mitosis[2]. Pitstop 2 (1-30 μM; 24 h) inhibits the growth of dividing cancer cells and causes apoptosis[2]. Pitstop 2 (1-30 μM; 48 h) has no effect on non-tumourigenic NIH3T3 fibroblasts' ability to proliferate and remain viable[2].

Cell Viability Assay[2]
Cell Types: HeLa cells
Tested Concentrations: 1, 3, 10, 30 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: decreased the total number of viable HeLa cells markedly in a dose-dependent manner.

[1]. Leishmania donovani Internalizes into Host Cells via Caveolin-mediated Endocytosis. Sci Rep. 2019 Sep 2; 9(1): 12636.

[2]. Inhibition of clathrin by pitstop 2 activates the spindle assembly checkpoint and induces cell death in dividing HeLa cancer cells. Mol Cancer. 2013 Jan 17; 12:4.

Leishmania donovani is an intracellular parasite that causes visceral leishmaniasis, a leading cause of death and disease worldwide. The host cell membrane serves as the gateway for Leishmania entry into the cell. Although studies have shown that multiple host cell membrane receptors are involved in the entry of Leishmania donovani into host cells, its endocytic pathway remains unclear. This study explored the endocytic pathway of Leishmania donovani into host macrophages using specific inhibitors targeting two major endocytic pathways: clathrin-mediated endocytosis and caveolin-mediated endocytosis. The results showed that the clathrin-mediated endocytosis inhibitor pitstop 2 did not affect the entry of Leishmania donovani proflagellates into host macrophages. Interestingly, treatment with genistein (an inhibitor of caveolin-mediated endocytosis) significantly reduced endocytosis. A similar trend of no flagellate load in host macrophages also supports this finding. These results indicate that Leishmania donovani utilizes caveolin-mediated endocytosis to enter host cells. Our findings provide new insights into the phagocytic mechanisms of Leishmania donovani into host cells and hold promise for developing novel therapies against Leishmaniasis infection. [1]

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Background: During metaphase of mitosis, clathrin stabilizes spindle kinematic (K) fibers. Many antimitotic compounds target microtubule dynamics. Pitstop 2™ is the first small molecule inhibitor of the terminal domain of clathrin to inhibit clathrin-mediated endocytosis. We investigated its effect on the second function of clathrin in mitosis.
Results: Pitstop 2 did not affect the recruitment of clathrin to the spindle, but disrupted its post-localization function. Pitstop 2 arrested HeLa cells in metaphase by disrupting the integrity of the mitotic spindle and activating spindle assembly checkpoints, with a phenotype similar to clathrin depletion and Aurora A kinase inhibition.
Conclusion: Therefore, Pitstop 2 is a novel tool for studying clathrin spindle dynamics. Pitstop 2 reduced the viability of dividing HeLa cells but did not affect dividing non-cancerous NIH3T3 cells, suggesting that clathrin may be a novel antimitotic drug target. [2]

C20H13BRN2O3S2

473.362821340561

471.955

1419320-73-2

Clathrin-IN-1;1332879-52-3

136246422

Yellow to brown solid powder

5.5

1

5

3

28

767

0

C1(C=CC=C2C=CC=CC=12)S(=O)(=O)NC1S/C(=C\C2C=CC(Br)=CC=2)/C(=O)N=1

CGDLWHGPJPVPDU-ATVHPVEESA-N

InChI=1S/C20H13BrN2O3S2/c21-15-10-8-13(9-11-15)12-17-19(24)22-20(27-17)23-28(25,26)18-7-3-5-14-4-1-2-6-16(14)18/h1-12H,(H,22,23,24)/b17-12-

(NZ)-N-[(5Z)-5-[(4-bromophenyl)methylidene]-4-oxo-1,3-thiazolidin-2-ylidene]naphthalene-1-sulfonamide

Pitstop 2; Clathrin-IN-1; (NZ)-N-[(5Z)-5-[(4-bromophenyl)methylidene]-4-oxo-1,3-thiazolidin-2-ylidene]naphthalene-1-sulfonamide; 1419093-54-1; (Z)-N-(5-(4-Bromobenzylidene)-4-oxo-4,5-dihydrothiazol-2-yl)naphthalene-1-sulfonamide; 1419320-73-2; 4g55; 1332879-52-3;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 62.5 mg/mL (132.03 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.28 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (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 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL 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.

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 (Please use freshly prepared in vivo formulations for optimal results.)