Pinometostat, tazemetostat, GSK2879552, decitabine, and anacardic acid were purchased from SelleckChem (Houston, TX). Trametinib was purchased from ChemieTek (Indianapolis, IN). For Western blot analysis, antibodies against DNMT1 (ab19905, Abcam, Cambridge, UK), p21 (#2947, Cell Signaling Technology, Danvers, MA), BIM (#2933, CST), and GAPDH (Millipore Sigma, Burlington, MA) were used. For immunohistochemistry analysis, antibodies against p21(#2947, CST) and BIM (ab32158, Abcam) were used. For siRNA studies, siRNAS for DNMT(#4390771, Ambion, Austin, TX) and Control (Santa Cruz Biotechnologies, Santa Cruz, CA) were used.
Pinometostat
Manufactured by Selleck Chemicals
Sourced in Italy, United States
Pinometostat is a laboratory equipment product used for biochemical research. It functions as a small-molecule inhibitor that selectively targets the epigenetic enzyme DOT1L, which is involved in the regulation of gene expression.
Automatically generated - may contain errors
Lab products found in correlation
Sgc0946, by Selleck Chemicals (2 mentions)
Ab32158, by Abcam (1 mentions)
Decitabine, by Selleck Chemicals (1 mentions)
Ab19905, by Abcam (1 mentions)
Anacardic acid, by Selleck Chemicals (1 mentions)
Trametinib, by Chemietek (1 mentions)
Tazemetostat, by Selleck Chemicals (1 mentions)
Gsk2879552, by Selleck Chemicals (1 mentions)
Gapdh, by Merck Group (1 mentions)
Panobinostat, by Merck Group (1 mentions)
Vorinostat, by Merck Group (1 mentions)
Celltiter blue, by Promega (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Envision multilabel reader, by PerkinElmer (1 mentions)
Dmem f12 with glutamax, by Thermo Fisher Scientific (1 mentions)
Rhfgf basic, by Promega (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Ab3594, by Abcam (1 mentions)
Ab7766, by Abcam (1 mentions)
Ab2886, by Abcam (1 mentions)
5 protocols using pinometostat
1
Molecular Mechanisms of Epigenetic Regulation
2
Cell Viability Assay with HDAC and DOT1L Inhibitors
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The HDAC inhibitors Panobinostat (Sigma) and Vorinostat (Sigma) and the DOT1L inhibitors Pinometostat (EPZ-5676) (Selleck Chemicals) and SGC-0946 (Selleck Chemicals), and DMSO (Sigma) were diluted in culture medium. First, the inhibitors were diluted in culture medium to a stock concentration that was twice as high as the highest concentration tested. The stock concentration was further diluted to obtain to the desired range of concentrations. Twenty-thousand cells were plated per well in a flat-bottom culture-treated 96 wells plate, in 50 µl culture medium and 50 µl culture medium with inhibitor was added. Four hours before measuring cell viability, Cell Titer Blue (Promega) was added to the wells and cells were incubated in the dark at 37°C under 5% CO2 conditions. After 4 hours, fluorescence was measured on the EnVision Multilabel Reader (Perkin Elmer). In order to calculate cell viability, values for fluorescence intensity of each condition were normalized against those of untreated cells. Each biological replicate represents an average of three technical replicates of an independent experiment.
3
Reprogramming hiF-T Cells to iPSCs
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We performed hiF-T reprogramming experiments as previously described (Cacchiarelli et al., 2015 (link); Mellis et al., 2021 (link)). Briefly, we expanded hiF-T cells in hiF-T GM without puromycin for one week. On day −1, we seeded CF-1 irradiated MEFs (Fisher #A34181) on 24-well plates (Corning #353047) coated with Attachment Factor (Fisher #S006100). On day 0, we seeded varying amounts (1–3 * 10^5) hiF-T cells per 24-well plate. On day 1, we began Yamanaka factor induction by switching media to hiF-T GM with 2 ug/mL doxycycline and without puromycin. On day 3, we switched media to KSR medium (KSRM): DMEM/F-12 with Glutamax (Life Tech #10585018) + 20% Knockout Serum Replacement (Life Tech #10828010) + 1X 2-mercaptoethanol (Life Tech #21985023) + 1X NEAA (Invitrogen #11140050) + P/S + 8 ng/mL rhFGF-basic (Promega #G5071) + 2 ug/mL doxycycline. We changed KSRM daily, and analyzed cells on day 21. We performed ≥2 biological replicates (i.e. different vials of hiF-T cells expanded and reprogrammed on different days with different batches of media) unless otherwise specified for all experiments. For reprogramming experiments with perturbations, we used the LSD1 inhibitor RN-1 (Millipore #489479) at a final concentration of 1uM and the DOT1L inhibitor pinometostat (Selleck Chemicals #S7062) at a final concentration of 4uM.
4
Investigating Epigenetic Regulation by DOT1L
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The following antibodies were used for immunoprecipitation and Western blot analyses: C-terminal anti-ERα (F-10 sc-8002, Santa Cruz Biotechnology, Dallas, Texas), rabbit anti-estrogen Receptor Alpha (ab32063, Abcam, Cambridge, UK), rabbit polyclonal anti-DOT1L (A300-953A, Bethyl Laboratories, Montgomery, Alabama), β-actin (A1978, Sigma Aldrich, Milan, Italy), Rabbit anti-KMT4/DOT1L (ab72454), anti-Histone H3, total, (ab1791), anti-H3K79me1 (ab2886), anti-H3k79me2 (ab3594), anti-H3K79me3 (ab2621), anti-H3K4me3 (ab7766), anti-H3K27me3 (ab24684) from Abcam, anti-Rabbit IgG Isotype Control (31235, Thermo-Fisher), and the anti-Mouse IgG antibody (RM104, Aurogene, Rome, Italy).
Cells were treated with the following compounds: DOT1L inhibitors EPZ004777 (S7353), Pinometostat (EPZ5676)(S7062), SGC0946 (S7079), all from Selleckchem, and with 4-hydroxytamoxifen (4-OHT) (H7904, Sigma-Aldrich), Fulvestrant (ICI 182,780) (I4409, Sigma-Aldrich), and β-estradiol (E887-5G, Sigma-Aldrich) or vehicles (DMSO and/or EtOH), according to different experimental settings.
Cells were treated with the following compounds: DOT1L inhibitors EPZ004777 (S7353), Pinometostat (EPZ5676)(S7062), SGC0946 (S7079), all from Selleckchem, and with 4-hydroxytamoxifen (4-OHT) (H7904, Sigma-Aldrich), Fulvestrant (ICI 182,780) (I4409, Sigma-Aldrich), and β-estradiol (E887-5G, Sigma-Aldrich) or vehicles (DMSO and/or EtOH), according to different experimental settings.
5
Pinometostat Dose-Dependent Cell Viability Assay
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Pinometostat (EPZ5676; Selleckchem, Houston, TX, USA, S7062) in DMSO was diluted in media to final concentrations of 10 nm , 100 nm , 1 μm, and 10 μm . The selected clones (pLX304 construct with V5 tag and MLL1‐ZC3H13 fusion) and vector and parental control were plated at 1000 cells/well in 96‐well plates along with media containing different concentrations of drug and 0.1% DMSO as control. The plates were incubated with drug for 3 days and resazurin assay was performed subsequently for cell viability and read in a spectrophotometer (Molecular Devices SpectraMax M5, Molecular Devices LLC., San Jose, CA, USA). Another group of 6‐well plates (initial seeding of 0.3 × 106 cell/well) treated with drug along with respective control was maintained for 15 days. On every third day, the cells were trypsinized, counted, and reseeded into new 6‐well (for drug treatment maintenance) and 96‐well plates (for resazurin assay after third day) along with media containing the respective drug concentration. The remaining cells were used for cell cycle analysis using flow cytometry as per the protocol described earlier (Daigle et al., 2013 ).
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