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Gant61

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GANT61 is a small molecule inhibitor that targets the GANT1 signaling pathway. It functions as an antagonist of the GLI transcription factors, which are key mediators of the Hedgehog signaling pathway.

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Lab products found in correlation

Mts assay, by Promega (2 mentions) Pierce agarose chip kit, by Thermo Fisher Scientific (1 mentions) Mitotane, by Selleck Chemicals (1 mentions) Osilodrostat, by Selleck Chemicals (1 mentions) Ketoconazole, by Selleck Chemicals (1 mentions) Roscovitine, by Selleck Chemicals (1 mentions) Oxaliplatin, by Merck Group (1 mentions) Ethidium homodimer 1, by Thermo Fisher Scientific (1 mentions) 5 fluorouracil, by Selleck Chemicals (1 mentions) Epirubicin, by Selleck Chemicals (1 mentions) Calcein am, by Thermo Fisher Scientific (1 mentions)

4 protocols using gant61

1

GLI-Mediated Regulation of PD-L1 Transcription

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In order to investigate GLI-bound PD-l1 transcription factor, chromatin immunoprecipitation (ChIP) assays were performed using the Thermo Scientific™ Pierce™ Agarose ChIP kit following the manufacturer's protocol. Briefly, MSI-H cells were treated without/with GLI-inhibitor GANT61 (Stemcell technologies) for 48 hrs, fixed with formaldehyde to crosslink and preserve protein-DNA interactions. Protein-DNA complexes were sheared using Micrococcal Nuclease digestion, followed by immunoprecipitation with ChIP-grade antibodies against GLI1 (R&D AF3324), GLI2 (R&D AF3526), positive (anti-RNA polymerase II antibody) and negative (Normal Rabbit IgG) control used for protein-bound DNA sequences. Crosslinking was reversed using NaCl and protein was digested using Proteinase K. GLI-bound PD-L1 expression were quantified using quantitative RT PCR with PD-L1 primer (Thermofisher Scientific).
Koh V., Chakrabarti J., Torvund M., Steele N., Hawkins J.A., Ito Y., Wang J., Helmrath M.A., Merchant J.L., Ahmed S., Shabbir A., Yan So J.B., Yong W.P, & Zavros Y. (2021). Hedgehog Transcriptional Effector GLI Mediates mTOR-Induced PD-L1 Expression in Gastric Cancer Organoids. Cancer letters, 518, 59-71.
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2

Pituitary Organoid Cytotoxicity Screening

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Organoids were grown in Matrigel™ domes within 96-well round-bottom culture plates. Recombinant human SHH was removed from the pituitary organoid growth media, 24 h prior to drug treatment. Organoids were treated with either vehicle (DMSO), cabergoline (Selleckchem S5842), ketoconazole (Selleckchem S1353), roscovitine (Selleckchem S1153), GANT61 (Stemcell Technologies 73692), pasireotide (TargetMol TP2207), mifeprostone (Selleckchem S2606), etomidate (Selleckchem S1329), mitotane (Selleckchem S1732), metyropane (Selleckchem S5416), or osilodrostat (Selleckchem S7456) at concentrations of 0, 1, 10, 100, 1000, and 10,000 nM, for 72 h. The percentage of cell viability was measured using an MTS assay (Promega G3580). Absorbance was measured at 490 nm and normalized to the vehicle. Concentrations were plotted in a logarithmic scale, and a nonlinear dose response curve regression was calculated using GraphPad Prism. An IC50 value for each drug treatment was determined based on the dose response curve, using GraphPad Prism analysis software.
Chakrabarti J., Pandey R., Churko J.M., Eschbacher J., Mallick S., Chen Y., Hermes B., Mallick P., Stansfield B.N., Pond K.W., Thorne C.A., Yuen K.C., Little A.S, & Zavros Y. (2022). Development of Human Pituitary Neuroendocrine Tumor Organoids to Facilitate Effective Targeted Treatments of Cushing’s Disease. Cells, 11(21), 3344.
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3

Activated GLI2 Signaling in Lgr5+ Cells

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All mouse studies were approved by the University of Cincinnati Institutional Animal Care and Use Committee (IACUC) that maintains an American Association of Assessment and Accreditation of Laboratory Animal Care (AAALAC) facility. We used a triple-transgenic model engineered to express an activated GLI2 allele, GLI2A, in Lgr5- expressing stem cells and their progeny in adult mice when treated with tamoxifen followed by doxycycline [16 (link)]. The triple-transgenic Lgr5-EGFP-IRES-CreERT2; R26-LSL-rtTA-IRES-EGFP; tetO-GLI2A mouse is abbreviated as iLgr5; GLI2A. ILgr5; GLI2A mice were treated with either vehicle (PBS, i.p., every alternate day), or GANT61 (50 mg/kg body weight, i.p., every alternate day; Stem Cell Technologies, 73692), after the completion of oral doxycycline treatment for 3 weeks. Stomach tissues were collected and analyzed for histology, immunostaining and qRT PCR.
Chakrabarti J., Holokai L., Syu L., Steele N.G., Chang J., Wang J., Ahmed S., Dlugosz A, & Zavros Y. (2018). Hedgehog signaling induces PD-L1 expression and tumor cell proliferation in gastric cancer. Oncotarget, 9(100), 37439-37457.
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4

Organoid-based Anticancer Drug Screening

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Human-derived gastric cancer organoids (huTGOs) were grown in 96 well plates and treated with either epirubicin (Selleckchem, S1223), oxaliplatin (Sigma-aldrich, 9512), or 5-Fluorouracil (Selleckchem, S1209) at concentrations of 0, 0.5, 1, 5, 10, 50, 100 and 200 μM or in combination with 5 μM GANT61 (Stemcell Technologies, 73692) for 48 hours. After 48 hours, organoid proliferation was measured using MTS Assay (Promega, G3580). The absorbance was measured at 495 nm, concentrations were transformed in logarithmic scale, and a best fit nonlinear dose-response curve was plotted using GraphPad Prism (GraphPad Software, San Diego, CA). huTGOs were also grown in 48 well tissue culture plates and treated with either DMSO, or, the combination of epirubicin, oxaliplatin, and 5-FU at their calculated IC50 concentration, or 5 μM GANT61 plus a combination of epirubicin, oxaliplatin, and 5-FU at their calculated EC50 concentration. The organoids were harvested at different time points (0, 24, 48 and 72 hrs), resuspended in 5% BSA in PBS and stained with calcein AM and Ethidium homodimer1 for 30 min, room temperature at dark (ThermoFisher Scientific, L3224). Labeled cells were analyzed using Canto-III flow cytometer. The average ratio of live/dead cells were calculated using FlowJo and plotted as mean ± SEM using GraphPad Prism software.
Chakrabarti J., Holokai L., Syu L., Steele N.G., Chang J., Wang J., Ahmed S., Dlugosz A, & Zavros Y. (2018). Hedgehog signaling induces PD-L1 expression and tumor cell proliferation in gastric cancer. Oncotarget, 9(100), 37439-37457.
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