Saddleback Journal of Biology - Saddleback College

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Fall 2009 Biology 3B Paper hepatolenticular degeneration (Scheinberg and Gitlin, 1952). There is a disruption of normal copper metabolism and copper deposition in tissues (Gitlin, 1975). To study the copper components in milk we used a mammary epithelial cell model, PMC-42 cells, grown on a Transwell® membrane as a monolayer with tight junctions. PMC-42 cells are very similar to normal breast epithelial tissue in cell composition and physiological responses (Krishnan and Cleary 1990). They are polarized and mimic the basal and apical sides of mammary epithelial cells. The basal (blood) side is where copper is delivered on different plasma proteins, such as albumin and transcuprein (Git et al., 2008). Medium is collected on the apical, or milk secretion side and we analyze the secretions. Mammary explants obtained from virgin female mice were also used as a model system to test for the effect of lactational hormone on CTR1 and ceruloplasmin. Based on findings from Whitehead et al. (1984), PMC42 cells have been shown to respond to hormonal stimuli as would normal breast epithelial tissue upon treatment with prolactin or stimulation of growth. Culture media were also analyzed after 48 and 96 hours post-incubation. We expect lactational hormones to increase the expression of CTR1 and ceruloplasmin at the mRNA level as well as at the protein level. Methods PMC 42 cell culture / Copper transport study PMC 42 cells were trypsinized and 100 ul of siCTR and non-targeting siRNA (as negative control) were added onto Transwells treated with Matrigel for cell polarization. The cells were incubated with RPMI containing 10% Fetal Bovine Serum at 37 C. Radioactive 64 Cu (5uM)–His (50uM) was delivered on the basal side 48-90h post transfection with siRNAs,. Apical and cellular secretions were counted with gamma counter. Quantifying expression of CTR1 mRNA CTR1 mRNA was isolated using RNABee solution and performing phenol chloroform extraction. The supernatant aqueous layer was collected, and isopropanol was added to it and allowed to sit overnight. cDNA was synthesized by mixing isolated RNA with reverse transcriptase, deoxynucleoside triphosphates, and random primer. The expression of CTR1 was quantified by performing Real Time PCR (50 cycles in triplicates with a fluorescent reporter probe), relating the expression to 18S ribosomal RNA as an internal control. Determining protein expression of CTR1knockdown siCTR (100nM) cells on the apical membrane were cut out of the Transwells. Five hundred ul of lysis buffer (10mM tris, 2% SDS, pH 7.2) was added, and the samples were incubated with shaking for 5 minutes. Five ul 4x sample treatment buffer was mixed with 20 ul of sample and then loaded into 12.5% acrylamide gel. Gel electrophoresis was performed with 25 ul loaded in each well, and the gel was transferred onto a membrane with transfer buffer (192 mM glycine, 25 mM Tris base, 150ml MeOH, 0.375g SDS, ddH2O). The membrane was blocked overnight with 5% dry milk (1g). Two ul of CTR1 primary antibody (rabbit anti-CTR1 from Jack Kaplan) was added afterwards and left to shake over night. It was then washed with TTBS 3 times (100ml 1x TBS + 100µL Tween 20) and 2 ul of CTR1 secondary antibody (Sigma monoclonol antirabbit antibody produced in mouse) was added and allowed to shake overnight. The membrane was washed twice with 1x TTBS. The membrane was then placed in developing solution [50 ml development buffer (pH 9.4), 500 ul BCIP + 500 ul NBT] in the dark and allowed to develop. After bands appeared, the membrane was placed in ddH2O. We expected Mammary Explants Mammary tissue from virgin female mice (6-8 weeks old) was harvested and sliced. They were then grown on wells plated with collagen and immersed in DMEM medium with and without lactational hormones (insulin, prolactin, dexamethazone). The wells were then incubated at 37 C for 96 hours. Secretions were collected from the apical side at 48 hours and 96 hours after incubation. Tissues that had been grown were weighed, and a 40% solution was made for each sample with SDS tank buffer (18g Tris base, 86.4g glycine, 60 ml 10% SDS, 30 ml 2% NaN3, H2O). The samples were then homogenized to prepare for gel electrophoresis. The concentrations of secretions were measured based on the Bradford protein assay. Two hundred ul of Bradford reagent was combined with water and BSA in multiples of 10 ul beginning at 0 ul and ending at 70 ul. The absorbance values at 595 nm were obtained with a spectrophotometer, and a standard curve was generated based on the known concentrations of BSA. Fifty ul of each sample was combined with 750 ul of water and 200 ul of Bradford reagent, where the absorbance at 595 nm was taken. Based on the absorbance values plotted against the standard curve, the concentrations were determined. Determining protein expression of CTR1 and CP from mammary explants Five ul 4x sample treatment buffer was mixed with 20 ul of sample (either from mammary tissue or secretions) and then loaded into 12.5% acrylamide gel (7% for ceruloplasmin). Gel electrophoresis was performed with 25 ul loaded in each well, and the gel was transferred onto a membrane with transfer buffer 65 Saddleback Journal of Biology Spring 2010
Fall 2009 Biology 3B Paper (192 mM glycine, 25 mM Tris base, 150ml MeOH, 0.375g SDS, ddH2O). The membrane was blocked overnight with 5% dry milk (1g). Depending on the study, 2 ul of CTR1 primary antibody (rabbit anti- CTR1 from Jack Kaplan) or 2 ul of CP primary antibody (Biomatic mouse CP AB) was added afterwards and left to shake over night. It was then washed with TTBS 3 times (100ml 1x TBS + 100µL Tween 20) and 2 ul of CTR1 secondary antibody (Sigma monoclonol antirabbit antibody produced in mouse) was added and allowed to shake overnight. The membrane was washed twice with 1x TTBS. The membrane was then placed in developing solution [50 ml development buffer (pH 9.4), 500 ul BCIP + 500 ul NBT] in the dark and allowed to develop. After bands appeared, the membrane was placed in ddH2O. Results CTR1 Knockdown: Effectiveness and Effect on Copper Uptake from Cu-His The CTR1 gene was knocked down using siRNA specific for CTR1 and compared to treatment with nonspecific siRNA. We were able to study the expression of CTR1 at the mRNA and protein level as well as the effect of CTR1 on copper uptake by mammary epithelial cells. There was no statistically significant difference in the amount of mRNA between siNeg with 1.13 million copies and siCTR 0.58 million copies (Figure 1). In Figure 2, there is no observable significant difference in the amount of CTR1 protein expression between siNeg and siCTR. Based on Figure 3, the counts per minute of radioactive Cu64 showed no statistical difference between apical and cellular readings treated with siNeg and siCTR. Figure 1 Relative expression of CTR1 mRNA after treatment of PMC42 cell monolayers (90h post transfection) with non-specific and CTR1-specific siRNA. The data represent means +/- SD for 5-6 determinations. Knockdown was with siCTR-1 (100nM). There was no statistically significant difference between the negative control and siCTR1. There was also no CTR1 knockdown at the mRNA level. 1 2 3 4 5 6 7 siNeg siCTR Figure 2 Samples from above were loaded onto a gel and transferred to a membrane in order to detect and observe protein expression. Negative control samples were loaded into lanes 2, 3, 4, and CTR knockdown samples were loaded in lanes 5, 6, 7. There was no significant difference in the amount of CTR1 protein expression. 66 Saddleback Journal of Biology Spring 2010
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TABLE OF CONTENTS Peer Reviewed Man
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Author(s) Title Page Angela C. Park
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