Stock concentrations of Azithromycin (20 μg/μL, AK-Scientific), CytD (2 mg/mL, Sigma Aldrich), Latrunculin B (2.5 mM, Tocris), Jasplakinolide (1 mM, Sigma Aldrich), and ML10 (10 mM, Lifearc) were made up in DMSO (Sigma Aldrich). Chloroquine (10 mM, Sigma Aldrich) and Heparin (100 mg/mL, Sigma Aldrich) were dissolved in H2O and RPMI, respectively. MMV291, S-MMV291, R-MMV291, S-W936, R-W936, S-W414, S-W415, and S-W827 (Walter and Eliza Hall Institute) were dissolved in DMSO to a 10-mM stock solution.
Latrunculin b
Manufactured by Bio-Techne
Sourced in United Kingdom, United States
Latrunculin B is a naturally occurring macrolide compound that acts as a potent inhibitor of actin polymerization. It binds to actin monomers, preventing their assembly into filaments. This disruption of the actin cytoskeleton can be useful for various cell biology and biochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Chloroquine, by Merck Group (2 mentions)
Y 27632, by Merck Group (2 mentions)
Verteporfin, by Merck Group (2 mentions)
Tgf β2, by R&D Systems (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Heparin, by Merck Group (1 mentions)
Jasplakinolide, by Merck Group (1 mentions)
Azithromycin, by AK Scientific (1 mentions)
Celllight bacmam 2, by Thermo Fisher Scientific (1 mentions)
Cytochalasin d, by Merck Group (1 mentions)
Staurosporine, by LC Laboratories (1 mentions)
U0126, by Promega (1 mentions)
4 protocols using latrunculin b
1
Preparation of Pharmacological Compounds
2
Quantifying NP Subcellular Localization in HT1080 Cells
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To quantify
subcellular localization of NPs in HT1080 cells, Rab7a-RFP and Lamp1-RFP
fusion constructs were expressed using a commercial baculovirus platform
(CellLight BacMam 2.0, Invitrogen), following manufacturing guidelines.
Pharmacological modulation of NP uptake was performed using the following:
staurosporine (1 μM; LC laboratories), latrunculin B (1 μM;
Tocris), cytochalasin D (1 μM; Sigma), chloroquine (50 μM;
Sigma), and ethylisopropyl amiloride EIPA (100 μM; Tocris).
Cells were rinsed in fresh media immediately prior to imaging; only
adherent cells were quantified. chloroquine dose–response measurements
were normalized to the C16proDOX control (rather than the
chloroquine-free control), in order to compare relative effects on
the potency of Pd-mediated C16proDOX activation itself.
50 μM chloroquine alone caused a 15–30% decrease in cell
viability.
subcellular localization of NPs in HT1080 cells, Rab7a-RFP and Lamp1-RFP
fusion constructs were expressed using a commercial baculovirus platform
(CellLight BacMam 2.0, Invitrogen), following manufacturing guidelines.
Pharmacological modulation of NP uptake was performed using the following:
staurosporine (1 μM; LC laboratories), latrunculin B (1 μM;
Tocris), cytochalasin D (1 μM; Sigma), chloroquine (50 μM;
Sigma), and ethylisopropyl amiloride EIPA (100 μM; Tocris).
Cells were rinsed in fresh media immediately prior to imaging; only
adherent cells were quantified. chloroquine dose–response measurements
were normalized to the C16proDOX control (rather than the
chloroquine-free control), in order to compare relative effects on
the potency of Pd-mediated C16proDOX activation itself.
50 μM chloroquine alone caused a 15–30% decrease in cell
viability.
3
HSC Cell Culture and Treatments
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HSC cells were seeded at 2 × 104 cells/cm2 atop premade ECM hydrogels and cultured in DMEM with 10% FBS and 1% PSG for 1 or 2 days. Then, HSC cells were cultured in serum-free DMEM with 1% PSG and subjected to the different treatments: TGFβ2 (2.5 ng/mL; R&D Systems, Minneapolis, MN, USA; for 3 days), the ROCK inhibitor Y27632 (10 µM; Sigma-Aldrich; for 3 days or 30 minutes only), the actin depolymerizer latrunculin B (10 µM; for 30 minutes only to preserve cell viability; Tocris Bioscience, Bristol, UK; ThermoFisher Scientific), or the YAP inhibitor verteporfin (0.5 µM; Sigma-Aldrich; for 3 days).
4
Modulation of HTM Cell Behavior
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HTM/GTM cells were seeded at 2 × 104 cells/cm2 on premade hydrogels/coverslips/tissue culture plates, and cultured in DMEM with 10% FBS and 1% PSG for 1 or 2 days. Then, HTM cells were cultured in serum-free DMEM with 1% PSG and subjected to the different treatments for 3 days: TGFβ2 (2.5 ng/ml; R&D Systems, Minneapolis, MN, United States), the ERK inhibitor U0126 (10 μM; Promega, Madison, WI, United States), the Rho-associated kinase (ROCK) inhibitor Y27632 (10 μM; Sigma-Aldrich), the actin de-polymerizer latrunculin B (10 μM; Tocris Bioscience; Thermo Fisher Scientific), or the YAP inhibitor verteporfin (0.5 μM; Sigma). The monolayer HTM cells were processed for immunoblot, qRT-PCR and immunocytochemistry analyses.
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