Mice bearing TBP-3743 tumors were randomized following tumor formation (Fig. E1 ) to receive combination BRAFi (vemurafenib, LC laboratories, Woburn, MA; 35 mg/kg in 150 μL of 1.5% methylcellulose/PBS, qdx8 via oral gavage) and aPDL1 (10F.9G2, bioXcell, West Lebanon, NH; 200 μg/mouse in 200 μL PBS, q2dx4 by intraperitoneal injection). A subset received aCSF1R (AFS98, bioXcell) equivalently dosed with aPDL1. Control received vehicle. 24h prior to animals being sacrificed, Macrin (10nmol of dye in 750 μg Macrin in 150 μL PBS) and/or DDNP (270 μg) were injected intravenously; lectin (100 μg in 100 μL) was injected intravenously 1h prior to animals being sacrificed. PBS cardiac perfusion was followed by 4% formaldehyde fixation overnight, and tissues were transferred to 10 mL CUBIC solution with 1 μg/mL 4’,6-diamidino-2-phenylindole (DAPI; Life Technologies, Carlsbad, CA), rocked for 48–72h at 37°C, and mounted in glass chambers in fresh CUBIC solution for imaging. DDNP uptake was compared to doxorubicin (LC laboratories; 15 mg/kg via intravenous injection in 200 μL PBS, λex/λem=470 nm/595 nm) or AF647-aPDL1 (200 μg), co-injected with either Macrin and/or lectin, 24h prior to animals being sacrificed.
Vemurafenib
Manufactured by LC Laboratories
Sourced in United States
Vemurafenib is a chemical compound used as a laboratory reagent for research purposes. It functions as a kinase inhibitor, specifically targeting the BRAF V600E mutation. The core function of Vemurafenib is to inhibit the activity of the mutated BRAF protein, which is involved in cellular signaling pathways.
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Lab products found in correlation
Trametinib, by LC Laboratories (6 mentions)
Trametinib, by Selleck Chemicals (2 mentions)
Erlotinib, by LC Laboratories (2 mentions)
Azd6244, by Selleck Chemicals (2 mentions)
Mycoalert kit, by Lonza (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Afs98, by BioXCell (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Crl 1619, by American Type Culture Collection (1 mentions)
Bupivacaine, by Merck Group (1 mentions)
Calpeptin, by Merck Group (1 mentions)
Ropivacaine, by Merck Group (1 mentions)
Veratridine, by R&D Systems (1 mentions)
Dacarbazine, by Selleck Chemicals (1 mentions)
Tetrodotoxin, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Osimertinib, by LC Laboratories (1 mentions)
Dimethyl sulfoxide (dmso), by Carl Roth (1 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
25 protocols using vemurafenib
1
Combination BRAF and PD-L1 Inhibition in Mice
2
Cell Viability Assay with MUH and Synergism Analysis
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4-Methylumbelliferyl heptanoate (MUH; 100 µg/ml) was used for the analysis of cell viability as previously described30 (link). The cells were treated for 3–5 days with vemurafenib (LC Laboratories, up to 20 µM), trametinib (Selleck Chemicals, up to 0.25 µM), B02 (Merck, up to 50µM31 (link)) or RI-1 (Sigma-Aldrich, up to 100 µM32 (link)) as indicated in the individual figures and figure legends. The equation: “log (inhibitor) vs. normalized response” was used for nonlinear regression analysis with the Graphpad Prism 6.0 software. The synergism analysis was performed using the following formula: Eexp = (Edrug1 + Edrug2) – (Edrug1 × Edrug2); Eexp: expected effect of the combined treatment; Edrug: Effect of a drug. The effect (E) represents the relative level of viability decline (0 < E < −1). The observed effect (Eobs) is the relative level of viability decline after the respective combined drug treatment (synergistic effect: Eexp < Eobs; additive effect: Eexp = Eobs; antagonistic: Eexp > Eobs).
3
Melanoma Xenograft Growth Assay in NSG Mice
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To assess melanoma growth in vivo, 1 × 106 melanoma cells (NF-1LOF: MeWo; BRAFMut: 451LU) in 100 µL PBS/Matrigel (1:1) were subcutaneously injected into the right flank of NOD scid gamma (NSG™; NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) mice. After formation of palpable tumors, mice were randomized into two (NF-1LOF, n = 7) or four (BRAFMut, n = 7 or n = 6) treatments groups, respectively, based on gender, age, and weight. PMD-026 (100 mg/kg in 10% dimethyl sulfoxide (DMSO) and 2% CremophorEL) or vehicle only was applied twice a day by oral gavage. The BRAFMut xenograft model additionally received a chemical additive diet containing 417 parts per million (ppm) vemurafenib (LC Laboratories; Woburn, MA, USA) or standard food ad libitum (ssniff Spezialdiäten GmbH; Soest, Germany). Treatment (NF-1LOF: 14 d; BRAFMut: 10 d) was prematurely stopped if tumors ulcerated or exceeded a volume of 1,000 mm3. Tumor size was monitored every other day by measurement of tumor length and width using a caliper and the tumor volume (V) was calculated employing the following formula: V = 0.4 × length × width2. All animal experiments were approved by the responsible regional authority (Regierungspräsidium Tübingen, AZ HT1/18).
4
Vemurafenib-Resistant Melanoma Cell Model
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Vemurafenib was purchased from LC Laboratories (Woburn, MA), and VERU-111 was synthesized as described previously (Figure 1A ). The human melanoma A375 cell line was acquired from ATCC (ATCC® CRL-1619) and maintained in DMEM with 10% FBS. Vemurafenib-resistant melanoma cells were built according to literature (Su et al., 2012 (link)). Briefly, cells were chronically selected by culturing A375 cells in increasing concentrations of Vem for at least 3 months and named VR1 cells. The isolated resistant VR1 cell line steadily increased IC50 values for Vem above 10 μm and maintained in full growth medium containing 5 μm Vem. VR1-SgSkp2 cells generated from VR1 cells and Skp2 was knocked out with guide RNA sequence: 5′-atgcacaggaagcacctcc-3′, screened with puromycin and maintained in full growth medium containing 5 μm Vem.
5
Melanoma Cell Line Experiments
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Human melanoma cell lines A375 and A431 (Cell Lines Service, Germany) were cultured in RPMI 1640 medium (Invitrogen, US) supplemented with 2 mM glutamine and 10% heat-inactivated fetal bovine serum (Gibco, US). Ropivacaine and bupivacaine (Sigma, US) were dissolved in water and lidocaine was reconstituted in Hanks Balanced Salt Solution. Veratridine (R&D Systems, US), vemurafenib (LC Laboratories, US), calpeptin (Sigma, US) and dacarbazine (Selleckchem, US) were reconstituted in dimethyl sulfoxide (DMSO). Tetrodotoxin (Sigma, US) was dissolved in citrate buffer.
6
Cell Culture Drug Preparation Protocol
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For cell culture studies, osimertinib (LC Laboratories, USA, Cat. No. 1421373-65-0), trametinib (LC Laboratories, USA, Cat. No. 871700-17-3), and vemurafenib (LC Laboratories, USA, Cat. No. 918504-65-1) were dissolved in dimethyl sulfoxide (DMSO) (Carl Roth, Germany, Cat. No. 4720.4) to a final stock concentration of 10 mM. Cisplatin (pharmacy of University Hospital of Cologne) was diluted to 3.33 mM in 0.9% NaCl.
7
Peptide-Mediated Modulation of STAT3 Signaling
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Two peptides, APTSTAT3-9R and APTSCR-9R, were custom synthesized by AnyGen (Gwangju, South Korea). Antibodies against the following proteins were used for western blotting: P-STAT3 (clone D3A7, catalog no. 9145; Cell Signaling Technology, Danvers, MA, USA); STAT3 (clone 124H6, catalog no. 9139; Cell Signaling); P-p44/42 MAPK (ERK1/2) (clone D13.14.4E, catalog no. 4370; Cell Signaling); p44/42 MAPK (ERK1/2) (clone 137F5, catalog no. 4695; Cell Signaling); and GAPDH (catalog no. MA5-15738; Invitrogen, Carlsbad, CA, USA). The antibody use for immune checkpoint blockade therapy was InVivoPlus anti-PD-1 (clone RMP1-14, catalog no. BP0146; BioXcell, Lebanon, NH, USA). Vemurafenib was purchased from LC Laboratories (Woburn, MA, USA).
8
Radioligand Binding Assay for CCK-8 and E2 17βG
9
Melanoma Cell Line Luciferase Assay
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A375 (RRID: CVCL_0132), K1735 (RRID: CVCL_F828), and Mel57 (RRID: CVCL_4454) cells expressing firefly luciferase and mCherry were cultured in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, l ‐glutamine, nonessential amino acids, sodium pyruvate, and MEM vitamins (all Life Technologies, Carlsbad, CA, USA). Mel57 cells were kindly provided by W. P. Leenders (Radboud University Medical Center, Nijmegen, the Netherlands), and K1735 and A375 were a gift from I. J. Fidler (MD Anderson Cancer Center, Houston, TX, USA). A375 and Mel57 cell lines have been authenticated by STR profiling using the GenePrint 10 system (Promega, Madison, WI, USA) and were cultured mycoplasma‐free, as confirmed by PCR. All cell lines were also tested negative for mouse pathogens by Impact I PCR profile (2) (IDEXX, Ludwigsburg, Germany). Vemurafenib was purchased from LC Laboratories (Woburn, MA, USA), Vemurafenib‐13C6 was obtained from the Slotervaart Hospital pharmacy, and elacridar was generously provided by GlaxoSmithKline (Research Triangle Park, NC, USA).
10
Establishing Melanoma PDX Models
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To develop PDX models, tumor fragments derived from metastatic melanoma, with approval by the local institutional review boards, were transplanted subcutaneously in sex-matched NSG mice (4–6 weeks old). One tumor fragment was implanted in each mouse. Tumors were measured with a caliper every 2 days, and tumor volumes were calculated using the formula (length × width2)/2. Tumors with volumes around 500 mm3 were randomly assigned into experimental groups. Special mouse diets were generated to reduce stress to animals by incorporating trametinib (LC Laboratories) into chow to achieve daily trametinib dosing at 3 or 5 mg/kg/day or combined vemurafenib (LC Laboratories) and trametinib dosing at 90 mg/kg/day and 0.7 mg/kg/day, respectively (Test Diet). DNA-PKi (NU7026, Selleckchem) was dissolved in saline (UCLA Division of Laboratory Animal Medicine pharmacy) and administered intraperitoneally at 6, 8, or 10 mg/kg/day. We derived model- or patient-matched vehicle-treated tumors and acquired trametinib-resistant tumors as plotted in Supplementary Fig. S1A. We also derived model-matched vehicle-treated tumors and early on-treatment tumors as plotted in Supplementary Fig. S6C and S6D.
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