Furin inhibitor 1, by Merck Group - Product details

1

Cell Viability Assay for 2D and 3D

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For the drug titration experiments, ~16,000 cells were seeded
into tissue-culture treated 96-well plates in RPMI 1640 growth media (2D
monolayers) or ultra-low attachment 96-well plates in RPMI 1640 growth median
with 0.75% methylcellulose. Cells were then grown for the next 72 hours in
presence of titrated inhibitors. At the 72 hr point, 1/10th volume of alamarBlue
reagent (ThermoFisher, DAL1100) was added to cells and incubated ~2 hours
for 2D monolayer cells and ~10 hours for 3D spheroids at 37°C.
Fluorescence signals were then measured in a fluorescence plate reader (TECAN,
#30016056, excitation at 560 nm, emission at 590 nm) to estimate relative number
of live cells at different dosages of the inhibitors. Wild type H23 cells were
used in the experiments where efficacies of small molecule inhibitors were
compared between 2D and 3D. To test whether CPD deletion sensitizes cells
against ARS-853, H23 cells with Safe-sgRNA and with CPD-sgRNA (no fluorescent
marker) were used. Small inhibitors were obtained from the following sources :
Savolitinib from Selleckchem (S7674), Linsitinib from VWR (# 10189–468),
FURIN inhibitor I from Sigma Aldrich (# 344930), and ARS-853 from Cayman
chemical (# 1629268–00-3).

Han K., Pierce S.E., Li A., Spees K., Anderson G.R., Seoane J.A., Lo Y.H., Dubreuil M., Olivas M., Kamber R.A., Wainberg M., Kostyrko K., Kelly M.R., Yousefi M., Simpkins S.W., Yao D., Lee K., Kuo C.J., Jackson P.K., Sweet-Cordero A., Kundaje A., Gentles A.J., Curtis C., Winslow M.M, & Bassik M.C. (2020). CRISPR screens in cancer spheroids identify 3D growth specific vulnerabilities. Nature, 580(7801), 136-141.

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2

Cell Viability Assay for 2D and 3D

Check if the same lab product or an alternative is used in the 5 most similar protocols

For the drug titration experiments, ~16,000 cells were seeded
into tissue-culture treated 96-well plates in RPMI 1640 growth media (2D
monolayers) or ultra-low attachment 96-well plates in RPMI 1640 growth median
with 0.75% methylcellulose. Cells were then grown for the next 72 hours in
presence of titrated inhibitors. At the 72 hr point, 1/10th volume of alamarBlue
reagent (ThermoFisher, DAL1100) was added to cells and incubated ~2 hours
for 2D monolayer cells and ~10 hours for 3D spheroids at 37°C.
Fluorescence signals were then measured in a fluorescence plate reader (TECAN,
#30016056, excitation at 560 nm, emission at 590 nm) to estimate relative number
of live cells at different dosages of the inhibitors. Wild type H23 cells were
used in the experiments where efficacies of small molecule inhibitors were
compared between 2D and 3D. To test whether CPD deletion sensitizes cells
against ARS-853, H23 cells with Safe-sgRNA and with CPD-sgRNA (no fluorescent
marker) were used. Small inhibitors were obtained from the following sources :
Savolitinib from Selleckchem (S7674), Linsitinib from VWR (# 10189–468),
FURIN inhibitor I from Sigma Aldrich (# 344930), and ARS-853 from Cayman
chemical (# 1629268–00-3).

Han K., Pierce S.E., Li A., Spees K., Anderson G.R., Seoane J.A., Lo Y.H., Dubreuil M., Olivas M., Kamber R.A., Wainberg M., Kostyrko K., Kelly M.R., Yousefi M., Simpkins S.W., Yao D., Lee K., Kuo C.J., Jackson P.K., Sweet-Cordero A., Kundaje A., Gentles A.J., Curtis C., Winslow M.M, & Bassik M.C. (2020). CRISPR screens in cancer spheroids identify 3D growth specific vulnerabilities. Nature, 580(7801), 136-141.

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3

Epigenetic Screening Compound Library Protocol

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The epigenetic screening compound Library (96-well format) was purchased from Cayman Chemical (item# 11076, batch# 0479267), each compound was provided in DMSO at a concentration of 10 mM. The library consists of 146 compounds from four major categories according to their enzyme targets, including 18 histone acetyltransferase (HAT) inhibitors, 55 histone deacetylase (HDAC) inhibitors, 32 histone lysine methyltransferase (KMT) inhibitors, 13 histone lysine demethylases (KDM) inhibitors, and 28 other epigenetic compounds. Compound hits UNC0642, CAY10433, and MS436 were repurchased from Cayman Chemical. BRD4 degrader dBET1 (cat# SML2687) and Furin inhibitor I (cat# 344930) were purchased from Sigma-Aldrich. BRD4 degrader MZ-1 (cat# S8889) and HBV replication inhibitor lamivudine (3TC) (cat# S1706) were purchased from Selleck Chemicals.

Yu X., Long Q., Shen S., Liu Z., Chandran J., Zhang J., Ding H., Zhang H., Cai D., Kim E.S., Huang Y, & Guo H. (2023). Screening of an epigenetic compound library identifies BRD4 as a potential antiviral target for hepatitis B virus covalently closed circular DNA transcription. Antiviral research, 211, 105552.

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4

Furin-Mediated Cleavage of ET-1 Peptide

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The full‐length fET‐1^CF peptide was cleaved with 20 U·mL⁻¹ furin (New England Biolabs, Ipswich, USA) overnight at room temperature yielding processed ET‐1^CF (pET‐1^CF). The reaction was terminated by the addition of 100 nm furin inhibitor I (Merck; 1 h incubation at RT) and subsequent flash freezing in liquid nitrogen. Peptides were stored at −20 °C until further use in binding assays.

Joedicke L., Trenker R., Langer J.D., Michel H, & Preu J. (2015). Cell‐free synthesis of isotopically labelled peptide ligands for the functional characterization of G protein‐coupled receptors. FEBS Open Bio, 6(1), 90-102.

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5

Chemical Characterization of PTPμ Regulation

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The following chemicals were purchased from EMD Millipore (San Diego, CA) and used at the concentrations indicated in parenthesis: ionomycin (5 µM), furin inhibitor I (30 µM), GM6001 (25 µM), DAPT (1 µM) and proprotein convertase inhibitor (PPCI, 25 µM). Calpain inhibitor I (ALLN) was purchased from Sigma-Aldrich (St. Louis, MO) and used at 20 µM. The serine protease inhibitors 3,4-Dicholoroisocoumarian (DCI), N-p-tosyl-L-phenylalanine ketone (TPCK) and aprotinin were purchased from Sigma and used at 100 µM, 25 µM and 10µg/ml, respectively. All inhibitors were made up in DMSO with the exception of calpain inhibitor I, which was made up in methanol. A methanol control behaved similarly to DMSO and was not included in the figures (data not shown). The SK18 monoclonal antibody, directed to the intracellular domain, and the BK2 monoclonal antibody, directed to the MAM domain of PTPμ, have been described previously [Brady-Kalnay et al., 1993 (link); Brady-Kalnay and Tonks, 1994 (link)]. Polyclonal antibodies to ADAM 10 and ADAM 17 were obtained from Calbiochem and Millipore, respectively. A monoclonal antibody to vinculin was obtained from Sigma-Aldrich.

Phillips-Mason P.J., Craig S.E, & Brady-Kalnay S.M. (2014). A protease storm cleaves a cell-cell adhesion molecule in cancer: multiple proteases converge to regulate PTPmu in glioma cells. Journal of cellular biochemistry, 115(9), 1609-1623.

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Furin inhibitor 1

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5 protocols using furin inhibitor 1

Cell Viability Assay for 2D and 3D

Cell Viability Assay for 2D and 3D

Epigenetic Screening Compound Library Protocol

Furin-Mediated Cleavage of ET-1 Peptide

Chemical Characterization of PTPμ Regulation

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