All drugs were suspended in vehicle control, dimethyl sulfoxide (DMSO; Corning). Compound C (CC; Millipore Sigma or MedChemExpress) was purchased in solution at a concentration of 10 mM in dimethyl sulfoxide (DMSO), stored at −80 or −20°C for less than 2 months prior to use, and used at a concentration of 5 μM for all experiments. Regorafenib and Sorafenib (MedChemExpress) were suspended in DMSO and used at a concentration of 2.17 μM unless otherwise indicated. Hoechst 33342 (Invitrogen) was purchased in solution at a concentration of 10 mg/ml and was diluted 1:2,000 in PBS for use. Puromycin (Millipore Sigma) was purchased as a powder, suspended to 10 mg/ml in water, and used at a final concentration of 1 μg/ml for all lentiviral transduction selections.
Regorafenib
Manufactured by MedChemExpress
Sourced in United States
Regorafenib is a multi-kinase inhibitor that targets various signaling pathways involved in tumor growth and angiogenesis. It is used as a research tool to study the effects of inhibiting these pathways in cell-based and animal models.
Automatically generated - may contain errors
Lab products found in correlation
Sorafenib, by MedChemExpress (3 mentions)
Sorafenib, by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Merck Group (1 mentions)
Compound c, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Corning (1 mentions)
Collagenase, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Phosphor akt, by Cell Signaling Technology (1 mentions)
Phosphor erk, by Cell Signaling Technology (1 mentions)
Phosphor jnk, by Cell Signaling Technology (1 mentions)
Stmn1, by Cell Signaling Technology (1 mentions)
Mk2206, by MedChemExpress (1 mentions)
Nanog, by Cell Signaling Technology (1 mentions)
Silencer select validated sirna, by Thermo Fisher Scientific (1 mentions)
Pd98059, by MedChemExpress (1 mentions)
Histone 3, by Cell Signaling Technology (1 mentions)
Mmp 9, by Cell Signaling Technology (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
13 protocols using regorafenib
1
Compound C and Regorafenib/Sorafenib Treatments
2
Modulating PASMC miR-15a-5p and VEGF
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Pulmonary artery tissue samples were incubated in Hanks' solution containing collagenase (1.5 mg/ml, Invitrogen; Thermo Fisher Scientific, Inc.) for 30 min at 4°C. The adventitia was carefully stripped off using fine forceps, and the endothelium was removed. The remaining smooth muscle was digested with collagenase and elastase for 50 min at 37°C. The PASMCs were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a 5% CO2 incubator. After growing to 70-80% confluence, 2×105 cells were transfected with miR-15a-5p (5′-TAA GGC ACG CGG TGA ATG CC-3′) and negative mimics (5′-CCC CCC CCC CCC CCC C-3′) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Following transfection for 4 h, a VEGF inhibitor (regorafenib, 4 nM, MedChemExpress) was added to the cells for 44 h at 37°C. Next, the cells (2×105 cells) were transfected with miR-15a-5p and VEGF plasmid for 48 h.
3
Gefitinib and Sorafenib Inhibit EMT
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Gefitinib was purchased from J&K Chemical (Beijing, China). Sorafenib, regorafenib, MK2206 (AKT inhibitor), and PD98059 (ERK inhibitor) were obtained from MedChem Express (Princeton, NJ, USA). The primary antibodies against Sox2, Oct4, Nanog, STMN1, E-cadherin, N-cadherin, vimentin, ERK, phosphor–ERK, Akt, phosphor–Akt, JNK, phosphor–JNK, MMP-9, Histone3, and β-actin were obtained from Cell Signaling Technology. The primary antibodies against FOXM1, E2F1, and MMP2 were purchased from Abcam. Silencer Select Validated siRNAs against STMN1 and FOXM1 were obtained from Life Technologies, Carlsbad, CA, USA.
4
Investigating Apoptosis Signaling Pathways
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Harmine (cat. no. HY-N0737A) was purchased from MedChemExpress and regorafenib (cat. no. A8236) was obtained by APeXBIO Technology LLC. The primary antibodies against cleaved-caspase-3 (cat. no. 9661S), cleaved-poly (ADP-ribose) polymerase (cleaved-PARP; cat. no. 9541S; 1:1,000) and DYRK1A (cat. no. 2771S; 1:1,000) were obtained from Cell Signaling Technology, Inc. The primary antibodies against PARP-1/2 (H-250) (cat. no. sc-7150; 1:500), anti-GAPDH antibody (FL-335) (cat. no. sc-25778; 1:500), myeloid cell leukemia-1 (Mcl-1) (S-19) (cat. no. sc-819; 1:500), phosphorylated (p)-AKT (1/2/3) (Ser473) (cat. no. sc-7985; 1:500) and caspase-3 (H-277) (cat. no. sc-7148; 1:500) were purchased from Santa Cruz Biotechnology, Inc. The primary antibody against α-tubulin (rabbit polyclonal antibody; cat. no. AF0001; 1:1,000) was obtained from Beyotime Institute of Biotechnology. The primary antibody against AKT (cat. no. 610836; 1:500) was purchased from BD Biosciences. The secondary antibodies, including DyLight™ 800 4X PEG-conjugated anti-rabbit-IgG (cat. no. 5151) and DyLight™ 800 4X PEG-conjugated anti-mouse-IgG (cat. no. 5470), were obtained from Cell Signaling Technology, Inc.
5
Cell Viability Assay with BBI608, Regorafenib, and HA15
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The WST-1 assay (Sigma-Aldrich, St. Louis, MO, USA) was used according to the manufacturer’s protocol to determine cell viability. The reagents, including BBI608, regorafenib, and HA15 (MedChemExpress, Monmouth Junction, NJ, USA) were used and added in HCT116 and HT29 cells for 48 h for cell viability detection.
6
Regorafenib and Cisplatin Induced ROS-Mediated Apoptosis
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Regorafenib and cisplatin were obtained from MedChemExpress (USA). N-Acety-l-Cysteine (NAC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). DCFH-DA ROS detection kit were obtained from Beyotime (Shanghai, China). Antibodies of eukaryotic initiation factor-2α (eIF2α, 9722S), phosphorylated (p)-eIF2α (3398S), caspase-3 (9662S), cleaved caspase-3 (9664S), activating transcription factor 4 (ATF4, 11815S), p-JNK (4668S), JNK (9252T), p-p38 (9211S), p38 (9212S), GAPDH (5174S) and HRP-conjugated secondary antibodies (7074) were purchased from Cell Signaling Technology (Danvers, USA). Antibodies of NOX5 (25350-1-AP) and Bcl-2 (60178-1-Ig) were purchased from Proteintech (Wuhan, China).
7
Validating IDH Mutant Compound Screens
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Drug screening was done using IDH inhibitors AGI-5198 (Agios) or BAY-1436032 (Bayer) or the FDA-approved Oncology Drug Set II library (National Cancer Institute) containing 107 compounds. The compounds Gemcitabine hydrochloride (Sigma-Aldrich), Paclitaxel (Sigma-Aldrich), Teniposide (Santa Cruz Biotechnology, Inc), Daunorubicin hydrochloride (SelleckChem), Romidepsin (MedChemExpress), Dactinomycin (BioViotica), Regorafenib (MedChemExpress), Omacetaxine mepesuccinate (Sigma-Aldrich), and Marizomib (Sigma-Aldrich), were selected for the validation studies. Initial NIH compound screens were carried out in a 96-well format on 7 IDH mutant cultures and validation studies were performed in 384-well plates on all 12 IDH mutant cultures using the STARlet automated pipetting system (Hamilton). Serial dilutions of the selected compounds were added after 24 hours and viability was assessed by CellTiter GLO 2.0 after five days. For details see Supplementary Methods .
8
Combination Therapy for Cancer Treatment
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ONC201 was administered orally in 10:70:20 DMSO:PBS:Cremphor El as described [4 (link)] and treated weekly at the indicated doses. Bevacizumab was procured from the Fox Chase Cancer Center pharmacy and diluted in PBS. Bevacizumab was administered through retro-orbital injections every other week at a dose of 5 mg/kg. Regorafenib was procured from MedChemExpress (HY-1031) and administered orally at 10 mg/kg per day dissolved in PBS for at least 22 days. Anti-murine VEGF-A inhibitory antibody (Biolegend 512,808) was administered at 10 micrograms by i.p. twice weekly. Mouse body weight was observed every 3 days and when body weight began to drop Regorafenib dosing was stopped.
9
Regorafenib-Induced Apoptosis in Cancer Cells
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Regorafenib was purchased from MedChemExpress (New Jersey, USA). Staurosporine (STS), N-acetyl-l-cysteine (NAC), ROS assay Kit, JC-1 assay Kit, and caspase-3/8/9 activity assay Kits were obtained from Beyotime (Shanghai, China). Rabbit monoclonal antibodies to PI3K (4292), Bim (2933), Bax (5023), Bak (12105), Puma (98672), Bad (9268), Bcl-xl (2764), p-AKT (Ser473) (4060), AKT (9272), FOXO3a (12829), Drp1 (8570), Mff (14739), MFN1 (14739), MFN2 (11925), and PARP (9532) antibodies were purchased from Cell Signaling Technology (Massachusetts, USA). Luciferase Mycoplasma Detection Kit, mouse monoclonal antibodies tubulin, actin and GAPDH antibodies were obtained from TransGen (Beijing, China). TurbofectTM transfection reagent and Mitotracker Deep Red 633 were obtained from Thermo Fisher Scientific (Massachusetts, USA). Cell counting Kit-8 (CCK-8) and Alexa Fluor® 488 annexin V/Dead Cell Apoptosis Kit were obtained from Dojindo (Kyushu, Japan). Hoechst 33258 probe and PI probe were purchased from Sigma-Aldrich (Missouri, USA). Enhanced chemiluminescence (ECL) was obtained from Biosharp (Beijing, China).
10
Nivolumab and Regorafenib in NSG Mice
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All mouse handling procedures were approved by the Institutional Animal Care and Use Committee. The vertebrate animals used in this study were female 4- to 6-week old NSG mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ, #005557) purchased from The Jackson laboratory. The mice were housed in a specific-pathogen barrier animal facility at the MD Anderson Department of Veterinary Medicine and Surgery. The veterinary care provided included feeding the animals acidified water, a Uniprim diet (Envigo, #TD.06596), and adequate crude protein, minerals, and vitamins.
Nivolumab (Opdivo, Bristol-Myers Squibb Company) was administered intraperitoneally every 5 days at 20 mg per kg body weight for 25 days. Regorafenib (#HY-10331, MedChemExpress LLC) was administered orally every day at 10 mg per kg body weight for 25 days.
Nivolumab (Opdivo, Bristol-Myers Squibb Company) was administered intraperitoneally every 5 days at 20 mg per kg body weight for 25 days. Regorafenib (#HY-10331, MedChemExpress LLC) was administered orally every day at 10 mg per kg body weight for 25 days.
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