Can you simplify these methods for me please. The methods are in bold. Please summarize all three methods please.
Cell Lines and Cell Culture. The ER-positiveHBC cell line MCF-7and the ER-negative HBC cell line MDA-MB-23 1 were kindly provided by Dr. Marc Lippman (Lombardi Cancer Center, Washington, DC). ER-negative HBC cell lines SKBR-3 and MDA-MB-435 were obtained from Dr. Steven Byers (Lombardi Cancer Center, Washington, DC) and Dr. Janet Price (Uni versity of Texas M. D. Anderson Cancer Center, Houston, TX), respectively. ER-negative HBC cell line MDA-MB-468 was kindly provided by Dr. Ann Hamburger (University of Maryland Cancer Center, Baltimore, MD). MDA MB-23l cells stably transfected with ER were as described previously (10). Cells were cultured routinely in DMEMIHam's F-12 medium (1:1) supple mented with 5% fetal bovine serum as described previously (5).
Retinoids and Growth Experiments. All-trans-RAwas purchasedfrom Sigma Chemical Co. TFAB, SR1 l003/Am580, and SRI 1262 were as de scribed previously (12). RA is an activator of RARa, RAR@, and RARy. TTAB activates the RARs only, whereas SRi 1003 is a selective activator of RARct. SR11262 is a selective activator of RARfJ/y. For growth experiments,cells were cultured as described above and treated with various retinoids for periods of 3, 6, and 9 days in the same medium. Control cells were treated with vehicle alone, and the medium and the retinoids were changed every 2 days. Cells were trypsinized and counted using a hemocytometer.
Southern and Northern Blot Analysis. Genomic DNA extractionsfrom various HBC cell lines, restriction digestion, and Southern blots were per formed according to standard procedures (1 3). RNA extraction and Northern blot analysis were performed essentially as described previously (14). The cDNA probes for hRARa exons 1 and 2 were as described previously (11). The plasmid containing the cDNA fragment of the human acidic ribosomal phosphoprotein P0 (clone p36B4) was as described previously (15). The cDNAs encoding human homologues of the krox proteins (egrl, egr2, and egr3; Ref. 16) were kindly provided by Dr. Vikas Sukhatme (Harvard Medical School, Boston, MA). The probe labeling was according to the random primer method of Feinberg and Vogelstein (17). The hybridization and washing conditions were as described by Sambrook et a!. (13). Promoter Deletional Constructs. RARa promot
Can you simplify these methods for me please. The methods are in bold. Please summarize all three methods please.
Cell Lines and Cell Culture. The ER-positiveHBC cell line MCF-7and the ER-negative HBC cell line MDA-MB-23 1 were kindly provided by Dr. Marc Lippman (Lombardi Cancer Center, Washington, DC). ER-negative HBC cell lines SKBR-3 and MDA-MB-435 were obtained from Dr. Steven Byers (Lombardi Cancer Center, Washington, DC) and Dr. Janet Price (Uni versity of Texas M. D. Anderson Cancer Center, Houston, TX), respectively. ER-negative HBC cell line MDA-MB-468 was kindly provided by Dr. Ann Hamburger (University of Maryland Cancer Center, Baltimore, MD). MDA MB-23l cells stably transfected with ER were as described previously (10). Cells were cultured routinely in DMEMIHam's F-12 medium (1:1) supple mented with 5% fetal bovine serum as described previously (5).
Retinoids and Growth Experiments. All-trans-RAwas purchasedfrom Sigma Chemical Co. TFAB, SR1 l003/Am580, and SRI 1262 were as de scribed previously (12). RA is an activator of RARa, RAR@, and RARy. TTAB activates the RARs only, whereas SRi 1003 is a selective activator of RARct. SR11262 is a selective activator of RARfJ/y. For growth experiments,cells were cultured as described above and treated with various retinoids for periods of 3, 6, and 9 days in the same medium. Control cells were treated with vehicle alone, and the medium and the retinoids were changed every 2 days. Cells were trypsinized and counted using a hemocytometer.
Southern and Northern Blot Analysis. Genomic DNA extractionsfrom various HBC cell lines, restriction digestion, and Southern blots were per formed according to standard procedures (1 3). RNA extraction and Northern blot analysis were performed essentially as described previously (14). The cDNA probes for hRARa exons 1 and 2 were as described previously (11). The plasmid containing the cDNA fragment of the human acidic ribosomal phosphoprotein P0 (clone p36B4) was as described previously (15). The cDNAs encoding human homologues of the krox proteins (egrl, egr2, and egr3; Ref. 16) were kindly provided by Dr. Vikas Sukhatme (Harvard Medical School, Boston, MA). The probe labeling was according to the random primer method of Feinberg and Vogelstein (17). The hybridization and washing conditions were as described by Sambrook et a!. (13). Promoter Deletional Constructs. RARa promot
