br Tissue Microarray Analysis br

Tissue Microarray Analysis
TMAs of breast tumor biopsies were obtained from the Manitoba Breast Tumour Bank (MBTB, University of Manitoba), which operates with the approval from the Faculty of Health Sciences, University of Manitoba, Research Ethics Board at the Bannatyne Campus. Collection, handling, and histopathological assessment of tumor tissues have been previously described [31,61]. The breast cancer TMAs were comprised of a cohort of 768 breast tumor biopsies/specimens (consisting of 447 ER− and 321 ER+ tumors). The estrogen receptor (ER) and progesterone receptor (PR) status was determined by the ligand binding assay (ER+ ≥3 fmol/mg protein, PR
+ ≥10 fmol/mg protein). The clinicopathological characteristics of the patient cohorts were provided by the MBTB and used for statistical analyses.
Immunohistochemistry
TMAs. Immunohistochemistry (IHC) was performed on TMAs using the methods as described previously [31,61]. Briefly, serial sections (5 μm) of the TMAs were stained with rabbit polyclonal Ethylmalonyl Coenzyme A to claudin 1 at a dilution of 1:150 (Life Technologies Inc., Burlington, ON, Canada), rabbit polyclonal PKCα C-20 (Santa Cruz
β actin
Figure 1. Endogenous claudin 1 protein levels in human breast cancer cell lines. Western blot analyses showing claudin 1 protein in a panel of human breast cancer cell lines. High levels of claudin 1 protein are shown in the T47D, MCF10A, and BT20 cell lines, whereas ZR75, MDA-MB231, and MCF7 cells displayed low levels of endogenous claudin 1. The nontumorigenic MDCKII cell line which exhibits high endogenous levels of claudin 1 was used as a positive control for claudin 1. β-Actin was used as a control for protein loading. ER+, estrogen receptor positive; ER−, estrogen receptor negative.
Figure 2. Subcellular localization of claudin 1 in human breast cancer cell lines. (A) IF microscopy was used to detect claudin 1 in breast cancer cell lines. The adheren junction protein E-cadherin was used as a positive control for membrane staining, and the MDCKII cells were used as a positive control for claudin 1 membrane localization. (B) Western blot analysis showing relative levels of claudin 1 in different subcellular fractions from two representative cell lines. SRA (steroid receptor RNA activator) and E-cadherin were used to confirm cytoplasmic (C) and membrane (M) fractions, respectively. Scale bars = 20μm.
B r> Figure 3. Immunohistochemical analysis of the claudin 1 protein in normal breast tissue and breast cancer. Representative claudin 1 staining in human invasive breast cancer, and healthy normal mammary ductal epithelial cells are shown. Tumor depicted in panel A shows both membrane (black arrows) and cytoplasmic staining (red arrows), while the tumor in panel B shows only cytoplasmic staining (red arrows). (C) Normal ductal epithelial cells in a healthy mammary gland tissue show high levels of claudin 1, while the basal-like myoepithelial cells (depicted by the arrows) are primarily negative. Scale bars = 20μm.
Biotechnology Inc.) at a dilution of 1:750, or the rabbit polyclonal PKCε C-20 (Santa Cruz Biotechnology Inc.) at a dilution of 1:750. The paraffin-embedded tissue sections were processed using the automated Leica BOND Rx system. Tissues were processed and incubated for 60 minutes with the primary antibody and 30 minutes with the secondary antibody following standard protocol. Validation of the claudin 1 antibody has been described previously [31]. The validation of the PKCα antibodies included using PKCα knockout mouse material, blocks provided by Michael Leitges, Biotechnology Centre of Oslo, University of Oslo [62].
from which we were able to retrieve interpretable data (intact, unfolded tumor sections) were considered for analysis. As well, of our 768 patient sample cohort, some tumor cores were unavailable for analysis due to exhaustion of the TMA. All IHC data were compiled into the database maintained by the MBTB and made available for correlation analyses and other statistical comparisons.
Quantification and Cutoff Selection. Positive staining of antibody was assessed by light microscopy. A semiquantitative assessment strategy of the stained tissues was employed as previously described [31,37]. Both staining intensity (scale 0-3) and the percentage of positive cells (0-100%) were multiplied to generate an H score ranging from 0 to 300. TMA staining was evaluated independently by two investigators (A.B. and C.P.). Where discordance (i.e., different
Fold regulation (log10) 
1 DMSO TPA
Figure 4. Activation of PKC by TPA in MCF7 cells led to the upregulation of claudin 1 expression and increased protein level in a time-dependent manner. Real-time PCR and Western blot analyses were carried out on MCF7 cells treated with TPA (100 nM TPA for up to 18 hours, as described in Materials and Methods). PKC activation led to (A) a large fold increase in claudin 1 transcript and (B) protein levels (n = 3 experimental replicates).