S., 3rd (2009) ErbB4 splice variations Cyt1 and Cyt2 differ by 16 amino acids and exert opposing effects around the mammary epithelium in vivo. been demonstrated to be necessary for differentiation of the mouse mammary gland (9, 11, 12). Interestingly, mice deficient of either or in their mammary epithelia exhibit a similar failure in the formation and differentiation of the milk-producing lobuloalveoli (3, 11, 12). The overlap of the phenotypes of gain and loss of function on ErbB4 expression and function in mammary epithelial cells and and that this process is necessary for the normal ErbB4-mediated differentiation of mammary gland epithelial cells. EXPERIMENTAL PROCEDURES Conditional Knock-out Mice and Immunohistochemistry Mice with mammary gland specific targeting of (line A) have been described earlier (3). Paraffin sections of the mammary glands from mice at pregnancy Rabbit Polyclonal to ATP5A1 day 18 or lactating day 1 were immunostained with the rabbit polyclonal antibodies anti-ErbB4 (sc-283; Santa Cruz Biotechnology) and anti-GLUT-1 (ab14683; Abcam). Immunohistochemical analysis was performed using HistomouseMax IHC staining kit (Invitrogen) following the manufacturer’s protocol. Cell Culture T-47D human breast cancer cells were maintained in RPMI supplemented with 10% FCS. HEK293 human embryonic kidney cells and MDA-MB-468 human breast cancer cells were maintained in DMEM supplemented with 10% FCS. Ligands and Inhibitors To stimulate or block ErbB4 signaling, cells were treated with 50 ng/ml NRG-1 (R&D Systems) or 10 m AG 1478 (Calbiochem), respectively. To stimulate HIF-1 with prolyl hydroxylase inhibitors, cells were treated for 20 h with 500 m dimethyloxallylglycine (DMOG; Cayman Chemicals) or 100, 200, or 400 m CoCl2 (Sigma-Aldrich). All treatments were carried out in the absence of serum. Expression Plasmids and Transfection All pcDNA3.1constructs have been described earlier (13,C15). Plasmids encoding wild-type or P402A/P564A double-mutant HIF-1 with HA tag were from Dr. William Kaelin (Addgene plasmids 18949 and 18955). STAT5a encoding plasmid pME18S-(16) and pGL3–(17) (kindly provided by Dr. Edith Pfitzner, Institute for Biomedical Research, Frankfurt, Germany) have been described earlier. pwas obtained from Clontech. Transient transfectants of HEK293 cells were generated using FuGENE 6 (Roche), and transient transfectants of T-47D cells were generated using Lipofectamine 2000 (Invitrogen) following the manufacturer’s recommendations. MDA-MB-468 cells were transduced with empty (pBABE-puro) or ErbB4 (pBABE-purotargeting siRNAs (#1, target sequence 5(isoform JM-a) (19), -(19), and (20) have been described earlier. Immunofluorescence Staining T-47D cells as well as MDA-MB-468 transfectants expressing ErbB4 JM-a CYT-1 were plated on glass coverslips and treated for 20 h in DMEM with or without 500 m DMOG followed by 30 min of treatment with or without 50 ng/ml NRG-1. After fixing with ice-cold methanol, the cells were washed with PBS and placed in blocking solution (5% goat serum and 0.01% Tween 20 in PBS) for 1 h at room temperature followed by overnight staining at 4 C with the anti-ErbB4 E200 diluted in blocking solution. After five washes with PBS, the secondary antibody goat anti-rabbit Alexa 488 (Invitrogen) and 0.5 g/ml DAPI (Sigma-Aldrich) diluted in blocking solution was applied for overnight incubation at 4 C. After five washes with PBS, the samples were mounted with Mowiol (Calbiochem). Samples were imaged with 7ACC1 a LSM510 META (Carl Zeiss) confocal microscope using plan-apochromat NA1.4 63 objective. One-micrometer-thick optical sections were imaged through the middle plane of the cells. The localization of the anti-ErbB4 epitope was analyzed from unprocessed images using FIJI ImageJ software (21). First, the cell was outlined right outside of the plasma membrane using segmented line tool, and fluorescence intensity was measured yielding total fluorescence intensity of the cell (promoter, HEK293 cells were plated on 24- or 96-well plates at a density of 1 1.5 104 cells/cm2. The following day, the cells 7ACC1 were transfected with 50 ng of empty pcDNA3.1 vector, pcDNA3.1together with 50 ng of pME18S-(Clontech) (plasmid quantities given for 24 wells). The day after transfection, cells were subjected to treatment with 0 or 200 m CoCl2 and/or 0 or 10 m AG 1478 for 24 h. Luciferase activity was measured using Bright-Glo luciferase assay (Promega) following the manufacturer’s protocol. The luminescence signal from the reporter plasmid was normalized by the fluorescence signal from the control plasmid. Chromatin Immunoprecipitation T-47D cells were starved overnight. 7ACC1