Anti occludin, Zymed® clone OCOC-3F10; anti Fas, BD Biosciences clone 13; anti ZO-1, Chemicon® MAB1520; anti FADD, USBiological clone 12E7; anti MUC1, Abcam inc. clone EP1024Y; anti-caspase 3, Cell Signaling Technology® (8G10); anti-caspase 8, Axxora® (1G12).
A breeding colony of occludin null homozygous mice is maintained in our animal facility. To derive occludin-null cells male and female hemizygotes are cross-bred and female homozygotes are selected and bred. These mice can lactate, although lactation performance is inconsistent (Monks, Webb and Neville, unpublished). For the current experiments, mice whose pregnancies were timed by the appearance of a vaginal plug, were sacrificed at day 15 of pregnancy and the 4th and 5th mammary glands excised and collagenase treated as described previously to obtain isolated epithelial organoids . These organoids are plated on FBS treated Lab Tek II, CC2 glass chamber slides (Nunc) where they form nearly confluent monolayers with well formed junctions as indicated by ZO1 staining within 3 to 6 days of culture (Wilson and Neville, data not shown). Organoids from wild type mice are handled similarly. These procedures have been approved by the Institutional Animal Care and Use Committee of the Anschutz Medical Campus of the University of Colorado Denver.
Cells and Cell culture
EpH4 mammary epithelial cells  were grown in DMEM (GibcoBRL, Grand Island, NY) with 10 mM HEPES (Sigma-Aldrich) and subcultured every 3-4 days . CIT3 mouse mammary epithelial cells were grown as described  in DMEM with Ham's F12 (50:50) supplemented with 2% heat-inactivated fetal bovine serum (FBS), 5 ng/ml epidermal growth factor (EGF), 10 mg/ml insulin, 100 U/ml penicillin and 100 mg/ml streptomycin. To differentiate CIT3 cells, the growth medium was modified by removal of EGF and addition of 3 mg/ml each of ovine prolactin (National Hormone and Pituitary Program, Rockville, MD) and hydrocortisone (Sigma Chemical Co. St. Louis, MO). MDCK cells were grown in MEM with 2 mM L-glutamine and Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, and 10% FBS . Cultures were maintained at 37°C in 5% CO2 in air. For primary cultures mammary epithelial cells from 15 day pregnant mice were isolated as described above.
For immunohistochemistry and measurement of transepithelial resistance, cells were trypsinized from polycarbonate flasks, plated at 1:2 and grown for 7 days, then plated at 2× confluent density on FBS treated Lab Tek II, CC2 glass chamber slides (Nunc) or Transwell® filters (Product #3413) as stated in figure legends. Cells were grown 3 days then, for CIT3 cells, switched to medium containing hormones for 2 days prior to the beginning of experiments.
Expression constructs and transient transfection
Mouse occludin ATCC #MGC-5797 was cut with BsaA1 in the second extracellular loop and Bcl1 in the 5' UTR and inserted in pFLAG-CMV-1 (Sigma-Aldrich), placing it downstream of a secretory signal peptide and N-terminal FLAG epitope tag (F-ΔOcc). As a control vector pFLAG-CMV-1-BAP, which encodes secretory, N-terminal FLAG tagged, bacterial alkaline phosphatase, was obtained from Sigma-Alrich. F-ΔOcc or pFLAG-CMV-1-BAP was transfected into cells using FuGENE® HD (Roche) according to the manufacturer's instructions. Cells were plated in the morning at confluent density, transfected in the evening, and transfection medium was replaced with differentiation medium the following morning.
Synthesis of linear CLYHYC was accomplished using Fmoc chemistry. Assembly of side-chain protected N-α-Fmoc amino acids (AAs) was carried out on a resin support. Acylation reactions were carried out for 2 hours using 3-fold excess of Fmoc-AAs activated with N, N'-diisopropylcarbodiimide (DIC) in the presence of N-hydroxybenzotriazole (HOBt). The N-terminal Fmoc group was removed with 20% piperidine in N,N-dimethylformamide. Linear peptide was cleaved from the fully dried peptide-bound resin with freshly prepared TFA solution (TFA 94%, water 2.5%, ethanedithiol 2.5%, triisopropylsilane 1%) for 2.5 hours under nitrogen atmosphere and then cyclized with 5% iodine in methanol and purified to 95% pure cyclic H-CLYHYC-OH by reverse phase HPLC. Peptide mass was confirmed by MALDI-TOF mass spectrometry (expected [M+H]+ 799.9, found 799.8). Cyclic H-CLYQYC-OH was made using the same process except that H was replaced by Q. Peptide mass was confirmed by mass spectrometry (expected [M+H]+ 790.9, found 790.6).
Activated caspase stains
Cells were live stained with Image-iT®LIVE Green Caspase 8 Detection Kit (Molecular Probes) and/or Image-iT®LIVE Red Caspase 3 and 7 Detection Kit (Molecular Probes). Stains were diluted into differentiation medium containing LYHY or LYQY. After 1 hr at 37° cells were rinsed in 4°C differentiation medium, and processed for immunofluorescence.
Caspase 9 and caspase 8/10 inhibition
Caspase 9 inhibitor (Z-LEHD-FMK Cat.#FMK008), 100 μM, caspase 8 inhibitor (Z-IETD-FMK Cat.#FMK007), 50 μM, or caspase 10 inhibitor (Z-AEVD-FMK Cat.#FMK009) (R&D systems), 50 μM, was dissolved in medium and incubated with cells for 2 h. LYHY peptide was then applied at 350 μM with inhibitors for 2 hours total. Caspase 3 staining was performed during the last hour of LYHY treatment as described above.
Cells were fixed in 2% paraformaldehyde, then permeabilized and blocked in 3% BSA in 0.1% Triton X-100 TBS. Antibodies were diluted into this solution and incubated with cells overnight at 4°C. In all cases secondary antibodies were goat anti host IgG, highly cross-absorbed, with appropriate fluors obtained from Molecular Probes.
TUNEL staining was performed using the Roche In Situ Cell Death Detection Kit, TMR red. Cells were fixed in 2% paraformaldehyde and permeabilized in 1% sodium citrate (tri-sodium salt) containing 0.1% TX-100. Staining was performed as per manufacturer's instructions.
Images were collected, processed and analyzed using SlideBook software (Intelligent Imaging Innovations, Inc.) on a Nikon Diaphot TMD microscope equipped for fluorescence with a Xenon lamp and filter wheels (Sutter Instruments), fluorescent filters (Chroma), cooled CCD camera (Cooke) and stepper motor (Intelligent Imaging Innovations, Inc.). Adjacent z-sections were collected and deconvolved using a nearest neighbor algorithm. Multi-fluor images were merged and renormalized.
Cells were washed with ice cold PBS before 0.5 ml of lysis buffer (30 mM Tris HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 2 mM EDTA, 5.7 μl/ml 100 mM PMSF, 20 μl/ml Roche Complete EDTA-Free Inhibitor Cocktail, and 1 μl/ml 1 M DTT) was added to the culture plate. Cells were scraped from the plate and placed in a microfuge tube. Cells in lysis buffer were constantly agitated for 20 minutes at room temperature and then spun at 13, 900 rpm for 20 minutes. The supernatant was collected in a fresh tube and stored at 4°C.
Lysates containing 500 μg protein were incubated with 10 μg mouse anti-FADD (BD Biosciences) antibody for 2.5 hours at room temperature. Protein A/G Agarose beads (20 μl, Santa Cruz) were added to the lysate/antibody mixture for 1 hour before being washed 3 times in lysis buffer, followed by a wash in high salt lysis buffer (regular lysis buffer with 500 mM NaCl), followed by a final wash in regular lysis buffer. Bead/antibody/FADD complex was resuspended in 50 μl 2 × Llaemmili buffer and boiled for 5 minutes. The sample was then spun at 2, 500 rpm for 5 minutes and the supernatant was collected in a fresh tube and stored at 4°C. Samples (30 μg original cell lysate, 25 μl IP product) were loaded on a 12% acrylamide gel and run at 100 Volts for about 1.5 hours. The gel was transferred onto PVDF and treated with rabbit anti-occludin antibody (1:2000, Santa Cruz) at 4°C overnight. The membrane was then treated with a donkey anti-rabbit IgG, Horseradish Peroxidase linked antibody (GE Healthcare).