1. Chemicals and antibodies
All chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA) unless indicated otherwise. Tiron was from Fluka (SchIrte, Deutschland). Ophiobolin A (OP-A) was from Adipogen (San Diego, CA, USA). cycloheximide (CHX), MitoTracker-Red (MTR), calcein-acetoxymethyl ester (calcein-AM), ethidium homodimer-1 (EthD-1), and 4',6-diamidino-2-phenylindole (DAPI), acetyl ester (CM-H2DCF-DA) was from Molecular probe (Eugene, OR, USA). Caspase inhibitors benzyloxy-carbonyl-Val-Ala-Asp-(OMe)-fluoromethyl ketone (z-VAD-fmk) was from R&D systems (Minneapolis, MN). Recombinant human TRAIL (the nontagged 19 kDa protein, amino acids 114-281) was from KOMA Biotech (Seoul, South Korea). The antibodies used in this study were anti-PARP, (Abcam, Cambridge, MA); caspase-3 (Stressgen, Ann Arbor, MI); α-tubulin, anti-ubiquitin, anti-ATF4 (Santa Cruz Biotechnology, Santa Cruz, CA); anti-phospho-eIF2α, anti-anti-phospho-eIF2α, anti-IRE1α, anti-PERK, anti-CHOP (Cell Signaling Technology, Beverly, MA); anti-COX II (Invitrogen, Carlsbad, CA); anti-PDI (Enzo Life Sciences, Farmingdale, NY, USA); rabbit IgG HRP, mouse IgG HRP, and goat IgG HRP (Molecular probe, Eugene, OR, USA).
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glucose DMEM supplemented with 10% fetal bovine serum (FBS) and 1%
antibiotics (GIBCO-BRL, Life Technologies, NY, USA). U2OS cells were cultured in high glucose DMEM supplemented with 10% FBS and 1% antibiotics.
3. Measurement of cellular viability (Live/Dead assay)
Cells were cultured in 24-well plates and treated as indicated. The test drugs were then removed and the cells were washed twice with PBS. For measurement of cellular viability, 2 μM calcein-AM, a green fluorescent indicator of the intracellular esterase activity of cells, and 4 μM EthD-1, a red fluorescent indicator of membrane damaged (dead) cells, were added to each well, and the plates were incubated for 5 min in 5% CO2 at 37°C. Cells were then observed under a fluorescence microscope (Axiovert 200M, Carl Zeiss, Germany) equipped with Zeiss filter sets #46 and #64HE. Viable cells, corresponding to those that exclusively exhibited green fluorescence, were counted in seven fields per well at 200 × magnification. Only exclusively green cells were counted as live because bicolored (green and red) cells cannot be unambiguously assigned to live or dead groups.
4. Examination of the stable cell lines expressing the fluorescence specifically in the endoplasmic reticulum
The stable T98G sublines expressing the fluorescence specifically in the ER were established by transfection of T98G cells with the pEYFP-ER vector (Clontech, Mountain View, CA, USA) and selection with fresh medium containing 500 µg/mL G418 (Calbiochem, Darmstadt, Germany).
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5. Western blotting
Cells were washed in PBS and lysed in boiling sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer (62.5 mM Tris [pH 6.8], 1% SDS, 10% glycerol, and 5% β-mercaptoethanol). The lysates were boiled for 5 min, separated by SDS–PAGE, and transferred to an Immobilon membrane (Millipore, Bredford, MA, USA). After blocking nonspecific binding sites for 30 min using 5% skim milk, membranes were incubated for 2 h with specific antibodies. Membranes were then washed three times with TBST and incubated further for 1 h with horseradish peroxidaseconjugated antirabbit, -mouse or -goat antibody. Visualization of protein bands was accomplished using ECL (Advansta, Menlo Park, CA, USA). The representative results from at least three independent experiments are shown.
6. Immunocytochemistry (ICC)
After treatments, cells were fixed with acetone/methanol (1:1) for 5 min at -20°C, blocked in 5% BSA in PBS for 30 min, and incubated overnight at 4°C with primary antibody [anti-COX II (1:500, mouse, Invitrogen, Carlsbad, CA), anti-PDI (1:500, rabbit, Enzo Life Sciences, Farmingdale, NY, USA), anti-CHOP (1:250, mouse, Cell Signaling Technology, Beverly, MA), anti-ubiquitin (1:1000, mouse, Santa Cruz biotechnology, Santa Cruz, CA)] diluted in antibody diluent solution (GBI Labs, Mukilteo, WA 98275, USA). And then slides were washed three times in PBS, incubated for 1 h at room temperature with anti-rabbit or anti-mouse Alexa Fluor 488 or 594 (1:1000, Molecular probe, Eugene, OR, USA). After, DAPI was stained for 5 min diluted in PBS (1:2000) and Slides were mounted with ProLong Gold antifade mounting reagent (Molecular probe, Eugene, OR, USA). Cell staining was visualized with a fluorescence microscope using (Axiovert 200M, Carl Zeiss, Germany).
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7. Gene silencing of CHOP (Knockdown experiments using siRNAs and shRNAs)
The siRNA duplexes used in this study were purchased from Invitrogen (Carlsbad, CA, USA) and have the following sequences: CHOP (NCBI accession no.
NM_004083,50-GAGCUCUGAUUGACCGAAUGGUGAA-30). Negative Universal Control (Invitrogen, Carlsbad, CA) was used as the control. After annealing of the pairs of siRNA oligonucleotide, cells in 24-well plates were transfected with 10 nM siRNA oligonucleotides using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions. For the knockdown experiments using CHOP-targeting shRNA, HEK293TN cells were transfected with the plasmid containing the non-targeting shRNA (SHC002V, Sigma-Aldrich, St Louis, MO) or the plasmid containing CHOP-targeting shRNA (TRCN0000364328, Sigma-Aldrich, St Louis, MO), together with pMD2.G (the envelope plasmid) and pPsAX2.0 plasmid (the packaging plasmid), using TransIT-2020 transfection reagents (Mirus Bio LLC, Madison, WI, USA) according to the manufacturer’s instructions. After 48 h of lentiviral particle production, T98G cells were infected with the filtered lentiviral medium (derived from HEK293TN cultures) supplemented with 10 μg/ml polybrene. To confirm successful siRNA- or shRNA-mediated knockdown, western blotting of the proteins of interest was performed.
8. Measurement of reactive oxygen species (ROS) production
CM-H2DCF-DA was used to measure the intracellular generation of hydrogen peroxide (H2O2). Briefly, 3 × 105 T98G cells were plated in 6-well plates and allowed to attach overnight. Cells were incubated with or without OP-A and then incubated with 5 μM of H2DCF-DA for 30 min in the dark at 37°C. After washing
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with Hank’s Buffered Salt Solution (HBSS) containing Ca2+ and Mg2+, cells were further processed for fluorescence activated cell sorting (FACS) analysis using a FACScan flow cytometer system (BD Biosciences, San Jose, CA). Data were analyzed using WinMDI 2.8 software (BD Biosciences, San Jose, CA).
9. Statistical analysis
Statistical analysis was performed with GraphPad Prism (GraphPad Software Inc., Sandiego, CA, USA) was used. Unless otherwise specified, each experiment was repeated at least three times. Normality of data was assessed by Kolmogorov-Smirnov testes and equal variance using Bartlett's test. For normal distribution, statistical differences were determined using an analysis of variance (ANOVA) followed by Bonferroni multiple comparison test. If the data were not normally distributed, Kruskal-Wallis test was performed followed by Dunn’s test. p <
0.001 was considered statistically significant.
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