We tested the inhibitory effect of the following compounds at the doses indicated except as otherwise stated, Tetrandrine (TETR 10 μM), Verapamil (VER 100 μM), Curcumin (CUR 75 μM), Bafilomycin (BAF 200 nM), Apilimod (APL 25 μM), Tamoxifen citrate (TMX 10 μM), Raloxifene hydrochloride (RLX 10 μM), Fulvestrant (ICI 182,780; FUL 100 μM) and β-Estradiol (EST 100 μM) were purchased from Sigma-Aldrich. Bapta-AM (BAP 10 μM) and YM201636 (YM 1 μM) and also controls Hydroxycloroquine (HCL 10 μM) endosomal acidification inhibitor and Teicoplanin (TEI 10 μM), an inhibitor of cathepsin L (Zhou et al., 2016 (link)) were purchased from Abcam. Stock solutions were dissolved in DMSO and working solutions were freshly prepared in DMEM 2% fetal bovine serum (FBS) at indicated concentrations. First, we pursued cell viability and cytotoxicity tests of all reagents using the CellTiter 96 Non-radioactive Cell Proliferation Assay (Promega) following the Manufacturer's instructions (Supplementary Fig. S2 , Supplementary Fig. S3 , Supplementary Fig. S4 ). We also studied the cytotoxic activity of the organic solvent DMSO. Based on these experiments we selected optimal non-toxic working concentrations for infection assays.
Apilimod
Manufactured by Merck Group
Apilimod is a laboratory equipment product developed by Merck Group. It is a small molecule that functions as a potent and selective inhibitor of the phosphoinositide-3-kinase (PI3K) signaling pathway. Apilimod targets the PI3K pathway, which is involved in various cellular processes and is often dysregulated in various disease states.
Automatically generated - may contain errors
Lab products found in correlation
Tetrandrine, by Merck Group (1 mentions)
Tamoxifen citrate, by Merck Group (1 mentions)
Raloxifene hydrochloride, by Merck Group (1 mentions)
Ym201636, by Abcam (1 mentions)
Bafilomycin, by Merck Group (1 mentions)
β estradiol, by Merck Group (1 mentions)
Celltiter 96 non radioactive cell proliferation assay, by Promega (1 mentions)
Curcumin, by Merck Group (1 mentions)
Verapamil, by Merck Group (1 mentions)
Fulvestrant, by Merck Group (1 mentions)
Bapta am, by Abcam (1 mentions)
Amiloride, by Merck Group (1 mentions)
Rifampin, by Merck Group (1 mentions)
Fk506, by Merck Group (1 mentions)
Torin1, by Merck Group (1 mentions)
Mts assay, by Abcam (1 mentions)
E coli lps, by Merck Group (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Thapsigargin, by Merck Group (1 mentions)
Ym201636, by Merck Group (1 mentions)
3 protocols using apilimod
1
Inhibitory Effect of Compounds on Infection
2
Screening Inhibitors of Macrophage Activation
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All commercially available inhibitors (bafilomycin A1, apilimod, Torin 1, thapsigargin, FK506, amiloride, rifampin) were from Sigma or Santa Cruz. MF4 and MF2 were kindly provided by Kevan Shokat, UCSF. All other compounds were provided by Celgene Corporation or synthesized at the Drug Discovery and Development Centre (H3D) at the University of Cape Town, South Africa. Inhibitors were prepared as 10 mM stocks in DMSO and tested at 10 μM final concentration (0.1% final DMSO). Where indicated, pure recombinant mouse IFNγ (final 10 ng/mL) (Roche) was added to BMDM cultures either together with inhibitors or 24 h prior to inhibitor treatment. Where indicated, E. coli LPS (Sigma) was added at 10 ng/mL. Each concentration of each agent was tested in triplicate in each experiment along with non-treated or vehicle (DMSO) treated controls. BMDM were observed daily. Forty-eight hours after addition of inhibitors, a 50 μL aliquot from each well was tested for nitrite and the rest frozen for ELISAs. Cellular viability was tested by the MTS assay (Abcam) according to the manufacturer’s instructions.
3
Inhibiting PIKfyve Regulates TGF-β1-Induced Fibroblast Contraction
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PIKfyve inhibitors Apilimod and YM-201,636 (Sigma) were dissolved at 10 mM in DMSO and arranged into a 10-step 3-fold serial dilution columnwise-wise in 3 rows each on a 384-well drug source plate. FLECSplates were pre-filled with 25 µL of serum-free DMEM, and then 100 nL of the PIKfyve inhibitor solutions were transferred from their source plate to the corresponding wells on the FLECSplate using a Biomek FXP pin tool. The FLECSplates were shaken at 100 RPM for min to ensure mixing. HLF cells were then seeded as previously described, with TGF-β1 already mixed into the cell suspension, into columns 2-23, while column was seeded with cells not receiving TGF-β1. Plates were imaged after 24 h as previously described. Percent inhibition was calculated by normalizing measured contraction values against both positive controls (e.g. no TGF-β1) and negative controls (TGF-β1 and vehicle only).
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