Cell culture and chemicals
Breast cancer cell lines BT474, MCF-7 and MDA-MB-361 were obtained from the American Type Culture Collection (Manassas, VA, USA). Human mammary epithelial cells (HMECs) were obtained from Cambrex (East Rutherford, NJ, USA). BT474 and MCF-7 cells were cultured in Dulbecco's modified Eagle's medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Hyclone) and 100 U/μl penicillin-streptomycin (Cellgro, Herndon, VA, USA); BT474 medium was also supplemented with ITS (insulin, transferring and selenium; Cellgro). MDA-MB-361 were cultured in RPMI-1640 (Hyclone) supplemented with 20% fetal bovine serum and 100 U/μl penicillin-streptomycin. HMECs were cultured in mammary epithelial growth medium (Cambrex). The PPARγ antagonist GW9662, the fatty acid palmitate, and the ceramide synthesis inhibitor fumonisin B1 were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Cell viability: proliferation assays
Cell viability after small hairpin RNA transfection or chemical treatments was assessed live cell counts after trypsinization and trypan blue staining using a hemocytometer. For high-throughput experiments, cells grown on 96-well plates were washed once with 1× phosphate-buffered saline (PBS), fixed with 2.5% formaldehyde, stained with Hoechst 33342 (Molecular Probes-Invitrogen, Carlsbad, CA, USA) and analyzed with an In Cell Analyzer 1000 (GE Healthcare, Piscataway, NJ, USA) high-content imaging system; cell counts and statistics were performed using In Cell Investgator 3.4 software (GE Healthcare).
Reverse transcription polymerase chain reaction
Total RNA was extracted from cells using TRizol (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized by reverse transcription of 2 μg of RNA in a 20 μl reaction using Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI, USA) at 42°C for 1 hour. PCR reactions were performed by using standard Taq polymerase (Fisher BioReagents, Fairlawn, NJ, USA) with the following primer pairs (forward and reverse, respectively): PPARγ, 5'-AGCCTCATGAAGAGCCTTCCA-3' and 5'-ACCCTTGCATCCTTCACAAGC-3'; fatty acid binding protein 4 (FABP4; aP2), 5'-GCATGGCCAAACCTAACATGAT-3' and 5'-CCTGGCCCAGTATGAAGGAAA-3'; hormone sensitive lipase (HSL), 5'-TACAAACGCAACGAGACAGGC-3' and 5'-TGTGATCCGCTCAAACTCAGC-3'; adipose tryglyceride lipase (ATGL), 5'-AGCTCATCCAGGCCAATGTCT-3' and 5'-GGTTGTCTGAAATGCCACCAT-3'; carnitine palmitoyltransferase 1 (CPT-1), 5'-TCACATTCAGGCAGCAAGAGC-3' and 5'-AATCGTGGATCCCAAAAGACG-3'; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-GCAAATTCCATGGCACCGT-3' and 5'-TCGCCCCACTTGATTTTGG-3'.
After the initial denaturation step (95°C for 3 minutes), PCR reactions consisted of 30 to 35 cycles of a 95°C step (15 seconds), a 52 to 55°C step (15 seconds), and a 72°C step (20 seconds), followed by a final elongation step at 72°C (5 minutes). PCR products were separated on 2% agarose-ethidium bromide gels. For quantitative determination of PCR product, a real-time reverse transcription PCR (RT-PCR) was performed on an ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA), using SYBR Green PCR Master Mix (Applied Biosystems). Primer pairs were the same as those used in regular RT-PCR. PCR reactions consisted of an initial incubation at 95°C (2.5 minutes) and 40 cycles of a 95°C step (15 seconds) and a 60°C step (60 seconds). Product levels were calculated after normalization with GAPDH or β-actin controls.
Immunofluoresence was performed on cells grown and treated either in 96-well plates or on cover slips in 24-well plates. In all cases, cells were fixed after treatment with 2.5% formaldehyde, washed with 1× PBS, permeabilized with 0.1% Triton-X 100 (Fisher Chemicals, Fairlawn, NJ, USA), blocked with 3% normal goat serum (Sigma-Aldrich), incubated with a 1:50 to 1:200 dilution of the primary antibody, washed with 1× PBS, incubated with a 1:800 dilution of the secondary antibody, washed again with 1× PBS, and finally stained with Hoechst 33342 (Molecular Probes-Invitrogen). Cells stained on 96-well plates were imaged using the In Cell Analyzer 1000 (GE Healthcare) and signal measurements and statistics were performed by the In Cell Investigator 3.4 software (GE Healthcare). Cells immunostained on cover slips were imaged by using a Leica TCS SP5 confocal microscope system (Leica Microsystems Inc., Bannockburn, IL, USA). Antibodies used were anti-activated Bax (6A7; BD Pharmingen, San Jose, CA, USA) and Alexa Fluor 568 goat anti-rabbit IgG (#A-11011; Invitrogen).
For detection of neutral fat stores, cells were grown on 96-well plates, fixed with 2.5% formaldehyde, washed with 1× PBS, stained with 10 μg/ml 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503; Molecular Probes), and counter-stained with Hoechst 33342 (Molecular Probes) for nuclei identification. Cells were imaged using the In Cell Analyzer 1000 and pictures were analyzed using the In Cell Investigator 3.4 software.
For fatty acid detection and quantification, total cellular lipids were extracted in accordance with a procedure described previously . Briefly, approximately 107 cells were pelleted and re-suspended in 3 ml chloroform:methanol (1:2) weight/0.05% butylated hydroxytoluene (BHT) to extract lipids. To monitor the recovery of fatty acids, 100 μg of heptadecanoic acid was added to each sample, before lipid extraction. Samples were centrifuged to remove cellular debris and chloroform and distilled water was added to form a biphasic solution. The two phases were separated by centrifugation and organic phases were transferred to a new tube and dried under nitrogen gas. Fats were then re-suspended in 250 μl toluene + 500 μl 1% sulfuric acid in methanol and incubated at 50°C overnight under nitrogen gas, followed by two steps of 1.25 ml 5% NaCl – 1.25 ml hexane extraction. Hexane extractions were combined, washed with 1.0 ml 2% NaHCO3, run through Na2SO4 columns, and dried under nitrogen gas. The fatty acid methyl esters were re-suspended in 1.0 ml methyl acetate and were analyzed by gas chromatography/mass spectrometry using an Agilent 6890 series gas chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a 5873 mass-selective detector. Part of the extraction was used for quantification of total and individual fatty acids under mass spectrometry and part for thin layer chromatography separation of triglycerides by a toluene-based system.
Reactive oxygen species assay
O2- generation was measured by using hydroethidine (Molecular Probes-Invitrogen, Carlsbad, CA, USA). Cells were incubated with a final concentration of 10 μmol/l of the dye for 30 minutes, and then fixed with 2.5% formaldehyde, stained with Hoechst 33342 and imaged with an In Cell Analyzer 1000. The hydroethidine signal was quantified using the In Cell Investigator 3.4 software.
The Student's two-tailed t-test was employed for the calculation of P values.