Tunicamycin (T7765, Sigma Aldrich) was reconstituted in DMSO to a concentration of 10 mg/ml. Cancer cell lines were plated and allowed to adhere to plates and reach ∼80% confluence. Cells were subsequently treated with Tunicamycin at a final concentration of 1 μg/ml for 48 h. Vehicle-treated cells received an equivalent volume of DMSO. When Tunicamycin treatment was performed with cycloheximide treatment, DLD1 and BT-20 cells were treated for 24 h with Tunicamycin prior to treatment with 75 μg/ml of cycloheximide (C-1189, AG Scientific) for up to 30 h. Following treatment, cells were washed three times in PBS and lysed in-well using RIPA buffer.
Cycloheximide (chx)
Manufactured by AG Scientific
Sourced in United States
Cycloheximide is a chemical compound commonly used in laboratory settings. It is a protein synthesis inhibitor that acts by blocking the translocation step in eukaryotic protein synthesis, thereby preventing the elongation of the polypeptide chain. Cycloheximide is widely utilized in various scientific applications that require the inhibition of protein synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Mg132, by AG Scientific (7 mentions)
Alexa fluor labeled secondary antibody, by Thermo Fisher Scientific (2 mentions)
N hexanoyl d erythro sphingosine d cer c6, by Matreya (2 mentions)
Sheep anti human tgn46, by Bio-Rad (2 mentions)
Chloroquine, by AG Scientific (2 mentions)
Leucine, by Merck Group (2 mentions)
Hela cell, by American Type Culture Collection (2 mentions)
Leucine free medium, by Atlanta Biologicals (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Actinomycin d, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Atlas Biologicals (2 mentions)
Actinomycin d, by AG Scientific (2 mentions)
Leucine, by AG Scientific (2 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (2 mentions)
Gefitinib, by Selleck Chemicals (1 mentions)
Tunicamycin, by Merck Group (1 mentions)
Mouse anti β actin clone ac 15, by Merck Group (1 mentions)
Mouse anti p230, by BD (1 mentions)
Mouse anti transferrin receptor, by Thermo Fisher Scientific (1 mentions)
14 protocols using cycloheximide (chx)
1
Tunicamycin-Induced ER Stress Assay
2
Serum and Leucine Starvation Response
3
Serum and Leucine Starvation of HeLa Cells
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HeLa cells (ATCC, Manassas, VA, Cat. # CCL-2) were maintained in culture at 37 °C in a humidified incubator with 5% CO2 in high glucose Dulbecco's modified Eagle's Medium (DMEM, Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (Atlas Biologicals, Fort Collins, CO) and 1% penicillin-streptomycin (Invitrogen). On the day of the experiment, cells were placed in serum-free medium (DMEM) or leucine-free medium (Atlanta Biologicals, Lawrenceville, GA) for 10 h. After 10 h of starvation, 10% serum or 0.76 mM leucine (Sigma, St. Louis, MO) was re-introduced into the culture medium for the indicated times. For experiments using Actinomycin D (Sigma) or cycloheximide (A.G. Scientific, San Diego, CA), cells were prepared as described above with the exception that either Actinomycin D (2 μg/mL) or cycloheximide (100 μg/mL) was added to the medium for 15 min or 2 min, respectively, prior to the re-introduction of serum or leucine.
4
Anticancer Effects of Daphne genkwa Extract
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The H1299 cell, human lung cancer cell, was provided by the Korean Cell Line Bank (Seoul, Korea). The cell line was cultured in RPMI 1640 mediun supplemented with 10% FBS and antibiotics-antimycotics (100 units/mL penicillin G sodium, 100 mg/mL streptomycin, and 250 ng/mL amphotericin B). The cells were incubated at 37°C and 5% CO2 in a humidified atmosphere.
YD (purity > 98.5%) was isolated and identified from a CHCl3-soluble fraction of the flower buds of Daphne genkwa, as described previously [24 (link)]. Cycloheximide (CHX) was purchased from A.G. Scientific (San Diego, CA, USA). Gefitinib was purchased from Selleckchem (Houston, TX, USA).
Antibodies against AXL and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
YD (purity > 98.5%) was isolated and identified from a CHCl3-soluble fraction of the flower buds of Daphne genkwa, as described previously [24 (link)]. Cycloheximide (CHX) was purchased from A.G. Scientific (San Diego, CA, USA). Gefitinib was purchased from Selleckchem (Houston, TX, USA).
Antibodies against AXL and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
5
Inhibition of Protein Synthesis and Degradation
6
Inhibition of Protein Synthesis and Degradation
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Cycloheximide (CHX) was purchased from A.G. Scientific, dissolved in ultra-pure water and used at final concentration of 50 µg/ml. MG132 was purchased from A.G. Scientific, dissolved in DMSO and utilized at final concentrations specified in figure legends.
7
Inhibitors for DNA Damage Response
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A list of all antibodies used in this study can be found in Supplementary Table 1 . The following reagents were used: ATM inhibitor KU59933 (Tocris), ATR inhibitor VE-831, DNA-PKs inhibitor KU57788 (NU7441), PARP inhibitor PJ34 (Selleckchem, Houston, TX, USA), SUMO inhibitor 2D-08 (Sigma, St. Louis, MO, USA), MG132, cycloheximide, and dimethyl sulfoxide (AG Scientific, San Diego, CA, USA).
8
Lipid and Antibody Reagent Protocol
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N-hexanoyl-d- erythro-sphingosine (d- cer-C6) and N-hexanoyl-l- erythro-sphingosine (l- cer-C6) obtained from Matreya were dissolved in pure ethanol (Merck) as stock solution. Cycloheximide was purchased from A.G. Scientific, rapamycin was obtained from EMD Millipore, and both were dissolved in DMSO as stock solutions. Sheep anti–human TGN46 was obtained from AbD Serotec. Goat anti-GRASP65 (C-20) was obtained from Santa Cruz Biotechnology, Inc. Mouse anti–β-actin (clone AC-15) was obtained from Sigma-Aldrich. Rabbit polyclonal antibody against GFP was purchased from Abcam. Mouse anti-p230, mouse anti-Sec31a, and mouse anti-EEA1 were obtained from BD. Mouse anti-M6PR (mannose-6 phosphate receptor) was obtained from Thermo Fisher Scientific, mouse anti–transferrin receptor was purchased from Invitrogen, and mouse anti-LAMP1 was obtained from Stressgen. Alexa Fluor–labeled secondary antibodies were obtained from Invitrogen, and HRP-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. Protein A–gold was obtained from the Department of Cell Biology at Utrecht University (Utrecht, Netherlands).
9
EGF and Alexa Fluor 555-EGF Experiments
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EGF was purchased from Sigma. Alexa Fluor 555-EGF was obtained from Invitrogen. Cycloheximide and MG132 were purchased from AG scientific (San Diego, CA, USA).
10
Transient Transfection and Protein Stability
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