For treated samples, human IFNalpha-2b (Intron A) was added to a final concentration of 1,000 IU/mL for 0, 6, 16, or 24 hours.
Growth protocol
U937T:PLZF45 cells, derived from human lymphoid cell line U937T stably expressing a construct for expression of full length PLZF under the control of a tetracycline regulated promoter (Ward JO, McConnell MJ, Carlile GW, Pandolfi PP, Licht JD, Freedman LP. The acute promyelocytic leukemia-associated protein, promyelocytic leukemia zinc finger, regulates 1,25-dihydroxyvitamin D(3)-induced monocytic differentiation of U937 cells through a physical interaction with vitamin D(3) receptor. Blood. Dec 1 2001;98(12):3290-3300) were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, Calif.), 1 mg/ml of G418 (Cellgro, Herndon,Va.), 0.5 ug/ml of puromycin (Sigma, St. Louis, Mo.), and 0.1 ug/ml of tetracycline (Roche, Indianapolis, Ind.). PLZF expression was induced by washing the cells twice in phosphate-buffered saline (PBS; Invitrogen) and replating them in the medium described above, with the omission of tetracycline.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted using Trizol reagent (Invitrogen) according to manufacturer's instructions. RNA integrity was verified by formaldehyde gel electropheresis.
Label
Cy3
Label protocol
Total RNA (20 g) was used to synthesize first-strand cDNA, which contains aminoallyl dUTP using random priming. The aa dUTP containing cDNA was conjugated to Cy3 (untreated) or Cy5 (treated) ester dye under basic conditions, and free nucleotide was removed by purifying the labeled cDNA with a GFX column (Amersham, Arlington Heights, IL).
For treated samples, human IFNalpha-2b (Intron A) was added to a final concentration of 1,000 IU/mL for 0, 6, 16, or 24 hours.
Growth protocol
U937T:PLZF45 cells, derived from human lymphoid cell line U937T stably expressing a construct for expression of full length PLZF under the control of a tetracycline regulated promoter (Ward JO, McConnell MJ, Carlile GW, Pandolfi PP, Licht JD, Freedman LP. The acute promyelocytic leukemia-associated protein, promyelocytic leukemia zinc finger, regulates 1,25-dihydroxyvitamin D(3)-induced monocytic differentiation of U937 cells through a physical interaction with vitamin D(3) receptor. Blood. Dec 1 2001;98(12):3290-3300) were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, Calif.), 1 mg/ml of G418 (Cellgro, Herndon,Va.), 0.5 ug/ml of puromycin (Sigma, St. Louis, Mo.), and 0.1 ug/ml of tetracycline (Roche, Indianapolis, Ind.). PLZF expression was induced by washing the cells twice in phosphate-buffered saline (PBS; Invitrogen) and replating them in the medium described above, with the omission of tetracycline.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted using Trizol reagent (Invitrogen) according to manufacturer's instructions. RNA integrity was verified by formaldehyde gel electropheresis.
Label
Cy5
Label protocol
Total RNA (20 g) was used to synthesize first-strand cDNA, which contains aminoallyl dUTP using random priming. The aa dUTP containing cDNA was conjugated to Cy3 (untreated) or Cy5 (treated) ester dye under basic conditions, and free nucleotide was removed by purifying the labeled cDNA with a GFX column (Amersham, Arlington Heights, IL).
Hybridization protocol
Hybridization was performed in slide hyb 3 hybridization buffer (Ambion, Austin, TX) beneath lifter slip coverslips (Erie Scientific, Portsmouth, NH) at 55°C for 16 h.
Scan protocol
Slides were washed for 5 min in each of three wash solutions containing 2x SSC/0.1% SDS, 2x SSC, and 0.2x SSC, centrifuged, and scanned using a GenePix 4000 scanner (Axon, Union City, CA).
Description
2004-02-04_PLZF45_+tet_16IFNcy5_0001_MIAME.gpr
Data processing
Using GeneSpring software version 5.0 (Silicon Genetics), the signal intensity ratio of the treated over the untreated sample was calculated using raw fluorescent intensities following local background correction. Only probes with fluorescent intensities of the untreated samples above 300 were used. The ratios were then normalized based on the distribution of all values with locally weighted polynomial regression (LOWESS).
