Temozolomide (#S1237, Selleckchem), cisplatin (#4333164, Teva Pharma), CPI-455 (#S6389, Selleckchem), IFNB1b (#I7662-14S, Biomol) were obtained from commercial vendors and stored as stock aliquots, as indicated by the manufacturers. We selected drug concentrations for chemotherapeutics (cisplatin; Temozolomide) based on physiologically relevant concentrations at each drug’s Cmax (the maximum tolerated serum concentration of each drug, drawn from published human studies)49 (link). We based concentrations for combinational exploratory compounds (CPI-455, IFNB1b) on primary literature and in-house in vitro testing of a 625-fold concentration range, optimized to induce a range of responses across classical NEN cell line spheroids (BON1, QGP1). Compounds were screened at equidistant 5-point, 625-fold concentration ranges using four technical replicates for long-term (168 h) chemotherapeutics screens or in equidistant 3-point, 625-fold concentration ranges with three technical replicates for short-term (24 h) combinational screens25 (link).
Cpi 455
Manufactured by Selleck Chemicals
Sourced in United States
CPI-455 is a laboratory equipment used for chemical processes. It is designed to facilitate precise control and monitoring of various parameters during chemical reactions or analyses.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Gsk j4, by Selleck Chemicals (2 mentions)
Temozolomide, by Selleck Chemicals (1 mentions)
Panobinostat, by Selleck Chemicals (1 mentions)
Vorinostat, by Selleck Chemicals (1 mentions)
Cytarabine, by Merck Group (1 mentions)
Vincristine, by Merck Group (1 mentions)
P cdk1 tyr15, by Cell Signaling Technology (1 mentions)
P chk1 ser345, by Cell Signaling Technology (1 mentions)
C myc, by Cell Signaling Technology (1 mentions)
Azd1775, by AstraZeneca (1 mentions)
Dab substrate kit, by Merck Group (1 mentions)
Countess 2 automated cell counter, by Thermo Fisher Scientific (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Mettl3, by Sangon (1 mentions)
Cisplatin, by Selleck Chemicals (1 mentions)
Kdoam 25, by Merck Group (1 mentions)
Potassium dichromate, by Merck Group (1 mentions)
Gsk343, by Selleck Chemicals (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
12 protocols using cpi 455
1
Optimized Chemotherapeutic and Combinational Screening
2
CPI-455 and Compound P Protocol
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CPI-455 (S8287; Selleckchem, Houston, TX, USA) was dissolved as 10 mmol/L of stock solution with DMSO (D5879; Sigma-Aldrich, St. Louis, MO, USA). CP was purchased from Sigma-Aldrich (479,306; Sigma-Aldrich) and was initially dissolved in PBS at a stock concentration of 3 mmol/L and further diluted in the medium used for cell and explant culture analysis.
3
AZD1775 Sensitivity in Leukemia Cells
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AZD1775 was provided by AstraZeneca (Wilmington, DE). The chemical structure of AZD1775 has previously been described (14 (link)). Cytarabine and vincristine were purchased from Sigma-Aldrich (St. Louis, MO) and diluted in water. Vorinostat, panobinostat, and CPI-455 were purchased from Selleckchem (Houston, TX). TP-0906 was purchased from MedChemExpress (Monmouth Junction, NJ). JQ1 was a kind gift from the laboratory of Dr. Jay Bradner. Antibodies specific to actin, c-MYC, pCDK1 Tyr15, CDK1, pCHK1 Ser345, PARP, γH2AX, histone 3 were purchased from Cell Signaling Technology (Danvers, MA). The antibody against KDM5A was purchased from Bethyl Laboratories (Montgomery, TX), and the antibody against trimethylated histone 3 lysine 4 was purchased from Active Motif (Carlsbad, CA). Primers to detect levels of AXL expression relative to GAPDH were 5′- CAGCGCAGCCTGCATGT-3′ (Forward) and 5′- TTGGCGTTATGGGCTTCG-3′ (Reverse).
4
Epigenetic Regulation of MCM2 and NUP37
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Anti-MCM2 antibody (HPA031496), anti-NUP37 antibody (ab220675), anti-GAPDH antibody (ab181602), Goat Anti-Rabbit IgG H&L (ab205718) and DAB Substrate Kit (ab64238) were purchased from Sigma and Abcam, respectively. Human MCM2, NUP37 and β-actin primer sets were purchased from Gene Chem (Shanghai, CN). Decitabine (S1200) and CPI-455 (S8287) were purchased from Selleck (Shanghai, CN). Dimethyl sulfoxide (DMSO) was purchased from Sigma.
5
STING Induction in Murine Cancer Cells
6
Cell Viability and Clonogenic Assays
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Cell viability was estimated using the WST assay (Sigma-Aldrich). For this purpose, cells were plated in 96 wells plate. After 24 h, cells were incubated with or without HU (Sigma-Aldrich) or CPI-455 (12.5 µM, Selleckchem). After 72 h of treatment, WST-1[2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] was added to the medium at a dilution of 1/10, followed by an incubation at 37°C for 2 h, before measuring the absorbance at 450 nm. Alternatively, cells were mixed with Trypan Blue and counted using the countess II automated cell counter (Life Technologies).
For clonogenic assay, U2OS cells were electroporated with siRNA as described before and seeded at 30 cells/cm2 in triplicates in six-well dishes. The day after, they were treated for 24 h with 50 µM HU or left untreated and then were allowed to grow for 10–15 days more before fixation and coloration with 1% Crystal Violet in H2O.
For clonogenic assay, U2OS cells were electroporated with siRNA as described before and seeded at 30 cells/cm2 in triplicates in six-well dishes. The day after, they were treated for 24 h with 50 µM HU or left untreated and then were allowed to grow for 10–15 days more before fixation and coloration with 1% Crystal Violet in H2O.
7
Jumonji Inhibitor Compound Preparation
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Chloroquine (Sigma-Aldrich) was
dissolved in H2O, whereas all other drugs were dissolved
in DMSO (Sigma-Aldrich). The Jumonji inhibitors used in this study
were obtained from Cayman Chemical, Ann Arbor, MI (GSK-J4, GSK-J5,
and CPI-455), Selleck Chemicals, Houston, TX (ML324), and Xcess Biosciences,
San Deigo, CA (KDM5-C70 and SD-70). JIB-04 E and Z isomers were synthesized
as previously described.23 (link) Compounds freshly
dissolved in DMSO were aliquoted to minimize freeze/thaw cycles and
stored at −20 °C.
dissolved in H2O, whereas all other drugs were dissolved
in DMSO (Sigma-Aldrich). The Jumonji inhibitors used in this study
were obtained from Cayman Chemical, Ann Arbor, MI (GSK-J4, GSK-J5,
and CPI-455), Selleck Chemicals, Houston, TX (ML324), and Xcess Biosciences,
San Deigo, CA (KDM5-C70 and SD-70). JIB-04 E and Z isomers were synthesized
as previously described.23 (link) Compounds freshly
dissolved in DMSO were aliquoted to minimize freeze/thaw cycles and
stored at −20 °C.
8
IFIT2, METTL3, YTHDF2, SETD1B Regulation
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Targeting human IFIT2 and non-targeting control small interfering RNAs (siRNAs) were purchased from RiboBio Co., Ltd. (Guangzhou, China). The METTL3, YTHDF2 and SETD1B short hairpin RNA (shRNA) sequence were purchased from Sangon Biotech Co., Ltd (Shanghai, China), and then ligated into pLVX-Puro-GFP empty vector to construct shRNA plasmid. The CPI-455 (#S8287) was purchased from Selleck Chemicals (Houston, TX, USA). The STM2457 (#T9060) was purchased from TOPSCIENCE Co. Ltd (Shanghai, China). All sequences for RNA interference and primers sequences used for the experiments were listed in Supplementary Table 4 .
9
Cytotoxicity Screening of Drug Compounds
10
STING Induction in Murine Cancer Cells
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Murine MC38 colon cancer cells, B16-F10 melanoma cells, 293T cells were cultured in DMEM medium. Murine CT26 colon cancer cells were cultured in RPMI-1640 medium. 10% FBS and 100 U/mL penicillin plus 100 μg/mL streptomycin were supplemented into all cell culture medium. All mediums and cell culture supplements were obtained from Gibco. All cells were cultured at 37 °C in a humid atmosphere containing 5% CO2. KDM5 inhibitor CPI-455 was purchased from Selleck. To induce STING expression, MC38, and CT26 were treated with 25 μM CPI-455 for 10 days.
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