All procedures were approved by the Institutional Animal Care and Use Committee at the University of Nice-Sophia Antipolis, Nice, France. SLC3A2 (CD98hc) conditional null mice, SLC3A2 fl/fl (Fe ´ral et al., 2007) were crossed with constitutively active FSP1Cre transgenic mice (The Jackson Laboratory, Bar Harbor, ME). All in vivo and ex vivo experiments, FSP1Cre, SLC3A2 fl/fl mice and age-matched SLC3A2 fl/fl (WT) littermate controls were used. Mouse line characterization is described in Supplementary Figure S1 and in the Supplementary Materials and Methods. For tape stripping, WT or KO young adult back skin was clipped and depilated using depilatory cream. Adhesive tape was applied and removed several times on the back skin of each mouse tested to remove the stratum corneum. Mice were sacrificed and back skin was collected and processed 7 days after tape stripping. Thickness of the epidermis was quantified using Image J software (National Institutes of Health, Bethesda, MD). For 12-O-tetradecanoylphorbol-13-acetate treatment, WT or KO young adult back skin was clipped and treated three times with 17 nmol 12-O-tetradecanoylphorbol-13-acetate (Sigma, St Louis, MO) for 1 week. Mice were sacrificed and back skin was collected and processed 1 hour after the last treatment.
12 o tetradecanoylphorbol 13 acetate
Manufactured by Merck Group
Sourced in United States, Germany
12-O-tetradecanoylphorbol-13-acetate is a chemical compound that is commonly used as a laboratory reagent. It serves as a potent activator of protein kinase C, a family of enzymes involved in various cellular signaling pathways. The compound is often utilized in scientific research to study the effects of protein kinase C activation on cellular processes and behavior.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Human melanocyte growth supplement, by Thermo Fisher Scientific (3 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (3 mentions)
Cholera toxin, by Merck Group (3 mentions)
Sodium butyrate, by Merck Group (3 mentions)
Indomethacin, by Merck Group (3 mentions)
Mushroom tyrosinase, by Merck Group (2 mentions)
Tween 20, by Merck Group (2 mentions)
7 12 dimethylbenz a anthracene, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Biosera (2 mentions)
Dmem high glucose, by Lonza (2 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (2 mentions)
Diphtheria toxin, by Merck Group (2 mentions)
Jsh 23, by Merck Group (2 mentions)
Grgdsp peptide, by Merck Group (2 mentions)
254cf medium, by Thermo Fisher Scientific (2 mentions)
Anti integrin αv blocking antibody, by Merck Group (2 mentions)
Fc soluble icam 1, by R&D Systems (2 mentions)
36 protocols using 12 o tetradecanoylphorbol 13 acetate
1
Epidermal Barrier Disruption in Mice
2
THP-1 Cell Line Differentiation and Fatty Acid Treatment
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The human monocytic leukemia cell line THP-1 was obtained from the American Type Culture Collection. THP-1 cells were maintained in RPMI-1640 growth medium (Invitrogen) supplemented with 10% FBS and 1% P/S at 37 °C under 5% CO2. Differentiation of THP-1 cells was induced by treatment with 10 ng ml−1 12-O-tetradecanoylphorbol-13-acetate (Sigma) in RPMI-1640 supplemented with 10% FBS and 1% P/S for 72 h. After 72 h of differentiation, the cells were used for the experiments. Before the incubations were performed, cells were kept in RPMI-1640 media containing 0.2% FAF-BSA overnight, and during the experiment, in RPMI-1640 media containing 2% FAF-BSA. The cells were then treated with RPMI-1640 media containing 2% FAF-BSA and 500 μM of BSA-complexed fatty acid for 24 h.
3
Fucoidan and L-DOPA Melanogenesis Regulation
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Fucoidan (from Fucus vesiculosus), 3,4-dihydroxyl-L-phenylalanine (L-DOPA), cholera toxin (CT), 12-O-tetradecanoylphorbol-13-acetate (TPA), and mushroom tyrosinase were from Sigma-Aldrich Co. (St. Louis, MO, USA). Antibodies specific for phospho-ERK1/2 (Thr202/Tyr204, #9101S), total (phosphorylated and non-phosphorylated) ERK1/2 (#9102), phospho-specific Akt (Ser473, #9271), total Akt (#9916), phospho-CREB (ser133, #9198), and total-CREB (#9197) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies for tyrosinase (C-19) and actin (I-19) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Microphthalmia Ab-1 (C5, MS-771-P0) was from NeoMarkers (Fremont, CA, USA). PD98059 was from Cell Signaling Technology. Secondary antibodies specific for anti-goat IgG (PI-9500), anti-mouse IgG (PI-2000), and anti-rabbit IgG (PI-1000) were purchased from Vector Laboratories (Burlingame, CA, USA).
4
Analyzing Cell Signaling Pathway Alterations
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The Sunset Yellow FCF was purchased from Hickson & Dadajee Private Limited in Mumbai. Fetal bovine serum (FBS) was purchased from Gibco, qualified, Brazil. Penicillin/streptomycin (Cat. No: 15240062: Thermo Fisher), Propidium Iodide (PI), 2′,7′ -dichlorodihydrofluorescein diacetate (DCFH2-DA) were purchased from Invitrogen (Carlsbad, CA, USA), JC-1 dye was procured from ENZO, Germany. Trypsin-EDTA solution (0.25%), phosphate buffers, and tween-20 were bought from Sigma Aldrich company (St. Louis, MO, USA). 7,12-dimethylbenz [a]-anthracene and 12-O-Tetradecanoylphorbol-13-acetate (TPA) were purchased by Sigma-Aldrich. The other chemicals (PCNA (10004805), λH2AX (D7T2V), EGFR (#2232 S), PEGFR (#2235 S), NRF2 (sc13032), GSK3β (#610202), and PGSKβ (sc-11757)) were all the highest purity grades and were purchased from Sigma-Aldrich unless otherwise stated.
5
Melanocyte and Melanoma Cell Line Maintenance
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Previously established RET-mice of line 304/B6 [4 (link), 5 (link)] were used. Normal human epithelial melanocytes (NHEM) were purchased from KURABO Co. and were maintained in melanocyte growth medium containing hydrocortisone and growth supplements. G361 cells were provided by Cell Resource Center for Biomedical Research, Tohoku University. Human SK-Mel28 melanoma cells and mouse B16 melanoma cell lines were purchased from the Riken Bioresource Center Cell Bank. The immortal melanocyte cell line melan-a was provided by the Wellcome Trust Cell Bank at St George's, University of London. This cell line was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 200 nM 12-o-tetradecanoyl phorbol-13-acetate (Sigma, USA). Other cell lines were maintained in DMEM supplemented with 10% FBS in a 5% CO2 atmosphere.
6
Cell culture conditions and treatments
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MCF-7 cells were grown in RPMI 1640 and COS-7 cells were grown in DMEM/High Glucose (Thermo Scientific). All media were supplemented with 10 % fetal bovine serum (Biosera), 100 IU/ml penicillin (Thermo Scientific) and 100 μg/ml streptomycin (Thermo Scientific). RPMI medium was additionally supplemented with 1 mM sodium pyruvate (PAA Laboratories). Cells were grown in 10 cm Petri dishes (Falcon) at 37 °C and 5 % CO2. When indicated, cells were treated with 16 nM 12-O-tetradecanoylphorbol-13-acetate (Sigma) or 2 μM GF109203X.
7
Maintenance of Diverse Cell Lines in Cell Culture
8
Biochemical Reagents for Cell Signaling
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DL-Dithiothreitol (DTT, D0632), N-Ethylmaleimide (NEM, E1271), Iodoacetamide (I1149), IGEPAL CA-630 (NP40, I3021), Triton X-100 (T9284), bovine serum albumin (BSA, A7906), Sodium dodecyl sulfate (SDS, L3771), Tween-20 (P9416), Ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA, E4884) and Trizma base (Tris, 93349), Sodium butyrate (NaBu, B5887) and 12-O-tetradecanoylphorbol-13-acetate (TPA, 4174) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Complete protease inhibitor cocktail (04693116001), phosphatase inhibitor cocktail (04906837001) and alkaline phosphatase (11097075001) were purchased from Roche Diagnostic (Mannheim, Germany). Ciprofloxacin (17850) was purchased from Fluka (Buchs, Switzerland). Poly(I:C) (LMW)/LyoVec (tlrl-picwlv) was purchased from Invivogen (Toulouse, France).
9
Cell Culture Reagents and Treatments
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Carvedilol, 3,4-dihydroxy-L-phenylalanine (L-DOPA), cholera toxin (CT), and 12-O-tetradecanoylphorbol-13-acetate (TPA) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Dulbecco’s Modified Eagle’s Medium (DMEM) and Dulbecco’s phosphate-buffered saline were purchased from WelGENE (Daegu, Korea). Fetal bovine serum (FBS), antibiotic-antimycotic, and trypsin-EDTA were purchased from Gibco (Grand Island, NY, USA). Medium 254 (Cascade Biologics, Portland, OR, USA) and FSK ([3R-(3α,4aβ,5β,6β,6aα,10α,10aβ,10bα)]-5-(Acetyloxy)-3-ethenyldodecahydro-6,10,10b-trihydroxy-3,4a,7,7,10a-pentamethyl-1H-naphtho[2,1-b]pyran-1-one) were purchased from Tocris Bioscience (Bristol, UK).
10
Melanoma and Melanocyte Cell Cultures
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B16-F0 (ATCC, CRL-6322), a murine melanoma cell line, was seeded in DMEM (Sigma-Aldrich, D2902) supplemented with 10% FBS (Corning, 35-015-CV) and antibiotic-antimycotic cocktail (GIBCO, 15240062), and incubated under an atmosphere of 5% CO2 at 37 °C. Melan-a (a gift from D. C. Bennet at St George's University of London), a murine melanocyte cell line, was cultured in RPMI 1640 (GIBCO, 31800089) supplemented with 10% FBS, antibiotic-antimycotic cocktail and 200 nM 12-O-tetradecanoylphorbol 13-acetate (Sigma-Aldrich, P8139) under an atmosphere of 5% CO2 at 37 °C.
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