DMEM (Dulbecco’s Modified Eagle Medium), FBS (fetal bovine serum), Collagenase Type I were purchased from Gibco (Life Technologies, Carlsbad, CA, USA). Restriction enzyme BglII and HindIII were bought from New England Biolabs (Ipswich, MA, USA). Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, CA, USA). Antibodies for C/EBPα, PPARγ and FABP4 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for HSL, p-HSL, ERK, p-ERK, S6 and p-S6 antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Methylisobutylxanthine, dexamethasone, Insulin, Oil Red O, Quinine hydrochloride dehydrate, Caffeine, Salicin, 6-propyl-2-thiouracil (PROP), Sucrose octaacetate, Hesperidin and Sodium Benzoate were bought from Sigma-Aldrich Co. (St. Louis, MO, USA). All tested bitter compounds were selected based on common knowledge and previous publications mentioned above. The bitter agonists were either dissolved in DPBS or DMSO, and final DMSO concentration did not exceed 0.1% (v/v) to avoid toxic effect on cells.
Methylisobutylxanthine
Manufactured by Merck Group
Sourced in United States
Methylisobutylxanthine is a laboratory chemical used as a reagent in various scientific applications. It is a xanthine derivative with a chemical formula of C₇H₁₀N₄O. This compound can be utilized in biochemical and cell culture experiments, but a detailed description of its core function is not available without the risk of extrapolation or interpretation.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Merck Group (26 mentions)
Insulin, by Merck Group (23 mentions)
Indomethacin, by Merck Group (16 mentions)
β glycerophosphate, by Merck Group (10 mentions)
Ascorbic acid, by Merck Group (8 mentions)
Oil red o, by Merck Group (7 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (7 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Alizarin red, by Merck Group (4 mentions)
Hydrocortisone, by Merck Group (4 mentions)
Alizarin red s, by Merck Group (3 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Insulin, by Thermo Fisher Scientific (2 mentions)
β glycerophosphoric acid, by Merck Group (2 mentions)
Ascorbate 2 phosphate, by Merck Group (2 mentions)
Alizarin red s a5533, by Merck Group (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions)
33 protocols using methylisobutylxanthine
1
Bitter Compound Screening in Adipocytes
2
Osteogenic and Adipogenic Differentiation of MSCs
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The WT and Alpl+/- MSCs were incubated with osteogenic medium (100 nmol⋅L−1 dexamethasone, 50 mg⋅mL−1 ascorbic acid and 1 mmol⋅L−1 b-glycerophosphate) (Sigma) for 21 d according to the manufacturer´s instructions. To assess osteogenic differentiation, the cells were fixed with 60% isopropanol and stained with 1% Alizarin Red (Sigma). The expression levels of Runx2 and OCN were assayed by western blotting on day 7 after the osteogenic induction.
The WT and Alpl+/- MSCs were cultured with adipogenic medium (0.5 mmol⋅L–1 methylisobutylxanthine, 0.5 mmol⋅L−1 hydrocortisone and 60 mmol⋅L–1 indomethacin; Sigma) for 14 d. The intracellular lipid accumulation was detected by staining with Oil Red O solution. PPARγ expression was assayed by western blotting on day 7 after the adipogenic induction.
The WT and Alpl+/- MSCs were cultured with adipogenic medium (0.5 mmol⋅L–1 methylisobutylxanthine, 0.5 mmol⋅L−1 hydrocortisone and 60 mmol⋅L–1 indomethacin; Sigma) for 14 d. The intracellular lipid accumulation was detected by staining with Oil Red O solution. PPARγ expression was assayed by western blotting on day 7 after the adipogenic induction.
3
Differentiation and Starvation of 3T3-L1 Adipocytes
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3T3-L1 cells were cultured in DMEM with 10% bovine calf serum (Sigma Aldrich, Saint Louis, MO) and 100 IU/ml penicillin/streptomycin (Invitrogen, Carlsbad, CA) until confluence. Two days after confluence, the cells were induced for differentiation with the differentiation cocktail containing 10% fetal bovine serum (JRH Biosciences, Inc., Lenexa, KS), 115 µg/ml methylisobutylxanthine (Sigma Aldrich, Saint Louis, MO), 1 µg/ml insulin (Sigma Aldrich, Saint Louis, MO), and 390 ng/ml dexamethasone (Sigma Aldrich, Saint Louis, MO). The differentiation cocktail was replaced with DMEM containing 10% fetal bovine serum, 100 IU/ml penicillin/streptomycin and 1 µg/ml insulin two days later. The cultures were continued for additional 6 days.On day 7 of differentiation, adipocytes were starved in DMEM with 1 mg/ml glucose and 0.5% bovine calf serum for 12 hours, followed by various treatments as described in the figure legends. Both conditioned media and cells were collected for protein or RNA detection.
4
Adipocyte Differentiation of 3T3-L1 Cells
5
Evaluation of Swertiamarin's Anti-inflammatory and Anti-adipogenic Effects
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The RAW264.7 (TIB-71; ATCC, Manassas, VA, USA) murine monocytic cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA, USA) containing 10% foetal bovine serum (FBS; Gibco) until the cells reached 90% confluence. Then, the cells were serum-starved for 6 h and incubated with 10 ng/mL lipopolysaccharide (LPS; Sigma-Aldrich) in the presence of swertiamarin for 16 h. The LPS-induced inflammatory signals were examined by western blotting.
3T3-L1 mouse pre-adipocytes (CL-173, ATCC) were cultured in DMEM supplemented with 10% FBS until confluent. At 2 days post-confluency, the medium was replaced with DMEM containing 0.5 mM methylisobutylxanthine (Sigma-Aldrich), 0.125 mM indomethacin (Sigma-Aldrich), and 1.0 µg/mL insulin (Sigma-Aldrich). After 2 days, the induced cells were cultured in maintenance medium (DMEM containing 10% FBS, 1.0 µg/mL insulin, and 1 nmol/L T3) for 5 days and treated with swertiamarin at the indicated concentrations for 48 h. Oil Red O staining and quantitative real-time polymerase chain reaction (qPCR) were performed to evaluate the lipid content of cells.
3T3-L1 mouse pre-adipocytes (CL-173, ATCC) were cultured in DMEM supplemented with 10% FBS until confluent. At 2 days post-confluency, the medium was replaced with DMEM containing 0.5 mM methylisobutylxanthine (Sigma-Aldrich), 0.125 mM indomethacin (Sigma-Aldrich), and 1.0 µg/mL insulin (Sigma-Aldrich). After 2 days, the induced cells were cultured in maintenance medium (DMEM containing 10% FBS, 1.0 µg/mL insulin, and 1 nmol/L T3) for 5 days and treated with swertiamarin at the indicated concentrations for 48 h. Oil Red O staining and quantitative real-time polymerase chain reaction (qPCR) were performed to evaluate the lipid content of cells.
6
Adipogenic Differentiation Protocol
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Cells were seeded into 24-well plates at a density of approximately 1 × 105 cells per well. After the cells reached 80% confluence, the medium was changed into standard adipogenic differentiation induction medium (0.5 mmol/L of methylisobutylxanthine (Sigma, USA), 1 mmol/L of dexamethasone, 10 μg/mL of insulin (Sigma, USA), and 200 μmol/L of indomethacin (Sigma, USA)) and then cultured for another 3 weeks. The induction medium was changed every 3 days. Finally, cells were stained with Oil red O staining solution and microscopically observed under phase contrast (Zeiss, Germany).
7
3T3-L1 and hMSC Adipocyte Differentiation
8
Adipogenic Differentiation of 3T3-L1 Fibroblasts
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Murine 3T3-L1 fibroblasts (ATCC®, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% FBS (Life Technologies, Waltham, MA, USA) and penicillin/streptomycin (Gibco). Cells were grown to confluency and differentiated into adipocytes using adipogenic medium supplemented with 0.5 mM methyl-isobutyl-xanthine (Sigma-Aldrich, company, city, country), 1 µM dexamethasone (Sigma-Aldrich), 10 µM insulin (Sigma-Aldrich), and 100 µM indomethacin (Sigma-Aldrich). After cultivation for 3 days, cells were switched to DMEM containing 10 μM insulin for 1 day (one cycle). Preadipocytes were transfected with 60 pmol of mouse PIAS1 siRNA (sc-36220) or control siRNA, either as fluorescein conjugate (sc-36869) or unconjugated (sc-37007; all Santa Cruz Biotechnology, Santa Cruz, CA, USA) using lipofectamine® RNAiMAX transfection reagent (Thermo Fisher Scientific; Waltham, MA, USA) and analyzed 48 h later. Cells were fixed in 10% formaldehyde solution (Roth), stained with 0.5% Oil red O (Sigma) and examined under an inverted light microscope (Motic AE31, Motic, Wetzlar, Germany).
9
Differentiation of 3T3-L1 Preadipocytes into Adipocytes
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3T3-L1 preadipocytes were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 100 IU/ml penicillin and 100μg/ml streptomycin at 37°C in humidified air containing 5% CO2. The 3T3-L1 cells were differentiated into adipocytes as described previously [28 ]. Briefly, they were grown to 90% confluence and treated with 0.5 mM methyl-isobutylxanthine (Sigma I17018), 1μM dexamethasone (Sigma D4902) and 5μg/ml insulin (Sigma I0516). After 2 days the media was replaced with DMEM supplemented with 10% FBS and 5μg/ml insulin. Thereafter, the media was replaced every two days for ∼10 days. Differentiated adipocytes were confirmed by Oil O Red staining of the lipid pools present in the adipocytes. Briefly, the cells were washed with PBS, fixed with 10% formalin in PBS for 2 min and washed with distilled water. The cells were then stained with Oil O Red (Sigma O0625) for 1 hour, washed twice with distilled water and examined under a lightmicroscope.
10
Differentiation of MEFs to Adipocytes
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Primary MEFs were differentiated to adipocytes using the protocol of Fei et al.71 (link) with minor modifications. In brief, 1 × 106 MEFs (passage 3 or 4) were plated in six-well plates in DMEM-10% supplemented with 0.5 mM methylisobutylxanthine (Sigma, I7018), 1 M dexamethasone (DEX), 10 g/ml insulin and 10 M troglitazone (Tocris Bioscience, 3114/10). This media was kept for 3 days, and then replaced with DMEM-10% for another 2 days. After 5 days in culture 4-OHT was added to the culture twice for Mof deletion. For downstream analysis, cells were harvested before differentiation (MEFs) and after differentiation (iAdipo) followed or not by Mof deletion.
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