Mice were treated with 2.5% (w/v) DSS (M.W. = 36,000–50,000 Da; MP Biomedicals) dissolved in drinking water for 7 or 8 consecutive days, and then mice were sacrificed. Ring1a inhibitor (PRT4165, 8 mg/kg, Sigma-Aldrich) or the same dose of dimethyl sulfoxide was intraperitoneally injected into mice daily from day −2 to day 3 during DSS-induced colitis model to evaluate the effect of Ring1aKO inhibitor on mouse colitis. Body weight and disease activity index (DAI) were assessed daily. DAI scores were calculated according to weight loss, stool consistency, and stool blood content/rectal bleeding, as previously described.23 (link)
Prt4165
Manufactured by Merck Group
PRT4165 is a laboratory instrument designed for precision liquid handling. The device is capable of accurately dispensing and aspirating small volumes of liquids, facilitating various laboratory workflows. Its core function is to provide consistent and reliable liquid transfer functionality for research and analytical applications.
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Lab products found in correlation
Dextran sulfate sodium (dss), by Merck Group (1 mentions)
Dextran sulfate sodium (dss), by MP Biomedicals (1 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (1 mentions)
Atplite assay, by PerkinElmer (1 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (1 mentions)
Umk57, by Aobious (1 mentions)
Reversine, by Cayman Chemical (1 mentions)
H 151, by InvivoGen (1 mentions)
Trichostatin a tsa, by Merck Group (1 mentions)
Rgfp966, by Merck Group (1 mentions)
Gsk343, by Merck Group (1 mentions)
8 protocols using prt4165
1
DSS-Induced Colitis: Ring1a Inhibitor
2
Toxicity Assay and Erythroid Differentiation
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Toxicity tests were performed to determine the tolerated concentration of the RING1 inhibitor PRT4165 (Sigma-Aldrich). To assess an influence on erythroid differentiation, CD34+ bone marrow cells cultured as described above were treated with the well-tolerated concentrations of 12.5μM PRT4165 or DMSO for two weeks and with 2 U/ml erythropoietin during the second week. Then, cells were counted and split. 1500 cells were plated into methylcellulose and the remaining viable cells were analyzed by flow cytometry.
3
Ewing's Sarcoma Cell Line Characterization
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The Ewing’s sarcoma cell lines A673, SK-ES1, and A4573, which carry the EWSR1-FLI1 translocation types I, II, and III, respectively, and the HEK293 cell line from human embryonic kidney infected with AgT from SV40 (293T), were cultured in RPMI 1640 media (Gibco) and supplemented with 10% fetal bovine serum, l -glutamine, and penicillin/streptomycin. Cells were cultured at 37°C with 5% CO2. The A673 and SK-ES1 cell lines harboring shCTRL and shRING1B with seq#1 and seq#2 as well as A673 cell line with doxycycline inducible knockdown of EWSR1-FLI1 were previously described (11 (link), 18 (link)). hpMSCs were isolated following published protocols (21 (link)). Ectopic expression of EWSR1-FLI1 3xFLAG C terminus in HeLa cells was induced with doxycycline (0.5 μg/ml) (34 (link)).
All experiments performed with AZD1152 were incubated 72 hours, with the exception of RNA expression assays that were incubated 24 hours. For IC50 calculations, A673, SK-ES1, A4573, and 293T cell lines were seeded at 2000 cells per well in 96-well culture plates. AZD1152 and PRT4165 (Sigma-Aldrich) was added to complete growth medium; after 72 hours, cells were subjected to the ATPlite assay (PerkinElmer), and measurements were performed using a Tecan plate reader. Inhibitory concentrations were calculated using OriginPro 9.0 software.
All experiments performed with AZD1152 were incubated 72 hours, with the exception of RNA expression assays that were incubated 24 hours. For IC50 calculations, A673, SK-ES1, A4573, and 293T cell lines were seeded at 2000 cells per well in 96-well culture plates. AZD1152 and PRT4165 (Sigma-Aldrich) was added to complete growth medium; after 72 hours, cells were subjected to the ATPlite assay (PerkinElmer), and measurements were performed using a Tecan plate reader. Inhibitory concentrations were calculated using OriginPro 9.0 software.
4
Two-Stage Murine Skin Carcinogenesis
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The UVB irradiation protocol was performed as previously described61 (link),62 (link), with UVM-28EL (UVP Ultraviolet Product, Thermo Fisher) light source. For acute UV irradiation, tails were treated with 200 mJ cm−2 three times, collected 2 days after the last irradiation and analysed. For in vivo tumourigenesis the DMBA-UVB two-stage-induced carcinogenesis protocol was used. Dorsal or tail skin was treated with 120 μg ml−1 of DMBA (Sigma-Aldrich). UVB irradiation (180 mJ cm−2) was started 10 days after and continued three times a week until the end point. As permitted by the ethics committee, tumours smaller than 1,200 mm3 were collected. Tumours were classified as SCC in situ or SCC according to the tumour architecture61 (link),62 (link). For tumourigenesis, 8-week-old female SKH-1 mice were treated with 50 μl of PRT4165 (3 mg ml−1) (Sigma-Aldrich) every other day three times and irradiated with 250 mJ cm−2, three times a week until the end point63 (link).
5
Inhibition of PRC1 Activity Assay
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Inhibition of PRC1 activity was performed to analyse the PRC-dependent and independent activities of RYBP in promoter assays. Sixteen hours after transfection of the required plasmids by CaPO4 method (detailed in §4.3), HEK293 cells were fed with growth media supplemented with 50 µM of PRC1 inhibitor, PRT4165 (PRT4165, Sigma, cat. no. NSC600157) as previously reported by Ismail et al. and Gracheva et al. [23 (link),24 (link)]. The cells were maintained with PRT4165 supplemented media for further 24 h after transfection and the cells were harvested for whole cell lysates. The cell lysates were then prepared for luciferase reporter assay as described in §4.4.
6
Compound Screening in Cell Cultures
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Cells were grown under regular conditions until they reached ~80% confluence. Culture media (RPMI) containing the following compounds dissolved in DMSO (Fisher BioReagents), or an equal volume of DMSO, were added to the cells; 50 μM PRT (PRT4165, Sigma-Aldrich), 1 μM EPZ (EPZ011989, Cayman Chemical), 5 μM GSK126 (Xcess Biosciences), 1 μM UMK57 (Aobious), 0.5 μM reversine (Cayman Chemical) and H-151 (Invivogen). For experiments with treatment extending beyond 2 h, culture media were supplemented with 10% FBS and replenished after the first 24 h, with the applicable compound. For the long-term PRT treatment, cells were passaged under regular conditions and fresh media (RPMI + 10% FBS) with 50 μM PRT or 0.1% DMSO, were added for 48 h prior to passaging the cells, and cells were passaged 4–6 times. Images of cells were obtained using bright field microscopy (Evos, Life Technologies).
7
Quantitative Analysis of Chromatin Marks
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The cells were grown on coverslips for 5 days, adding Doxycycline (1 μl/ml), in a second set (per construct/per mark) in addition to Doxycycline, PRT4165 (Sigma-Aldrich) was added, different concentrations were tested (25, 40, 45 and 50 μM) and the 45 μM concentration was chosen. In a previous work in the laboratory, PRT4165 used in this range of concentrations had led to a reduction of ~50% of protein levels, demonstrated by western blot (18 (link)). The cells were then fixed and IF FISH for UbH2A, SMCHD1, H2K27me3 and MACROH2A was done as already described. The analysis of 30 cells by construct/mark was also blind.
8
Inducible XIST Expression in HT1080 Cell Line
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The HT1080 (male brosarcoma) cell lines with doxycycline inducible full length XIST cDNA constructs integrated into chromosome 8p have been described in previous publications (16, 17) . The XIST CRISPRmediated deletion constructs studied throughout this work were all created by targeted excision of speci c sequences from the Full length inducible XIST cDNA construct as described previously (13) . The additional deletion constructs Delta P MI, Exon 1, and Delta Delta, were generated through independent integration of partial XIST sequences into an 8p FRT integration site in the HT1080 cell line (17) . All XIST inducible constructs were under the control of a CMV promoter with a Tet repressor element as described previously (13, 17) . Induction of XIST was performed with 1µg/ml doxycycline, refreshed daily. All of the chemical inhibitors used in this assay were rst dissolved in DMSO and then added to culture media at the desired concentration. Inhibition of HDAC3 was performed using the chemical RGFP966 (Sigma-Aldrich) and the inhibition of all HDACs non-speci cally was performed using Trichostatin A (TSA;
Sigma-Aldrich). Inhibition of the catalytic activity of KMT5A (also known as PR-SET7/SET8/SETD8) was performed using the chemical ryuvidine from Abcam. Inhibition of PRC2 and PRC1 was performed using GSK343 and PRT4165 (both from Sigma-Aldrich) as described previously (13) .
Sigma-Aldrich). Inhibition of the catalytic activity of KMT5A (also known as PR-SET7/SET8/SETD8) was performed using the chemical ryuvidine from Abcam. Inhibition of PRC2 and PRC1 was performed using GSK343 and PRT4165 (both from Sigma-Aldrich) as described previously (13) .
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