Solutions (concentrations in mmole/L): Zeroing Bathing Solution (ZBS)
K+/Asp-(120), KCl (20), MgCl2 (4), NaCl (10), EGTA−/K+ (10), HEPES− (10), buffered with K+ to pH:7.4. Pipette Filling Solution (PFS): KCl (153), MgCl2 (4), CaCl2 (1), GdCl3 (0.2), HEPES− (10) buffered with K+ to pH: 7.4. Neutral buffered formalin (NBF): 10 % formalin, PO4− (75) buffered with Na+ to pH:7.0.
MCF-7 cell line was obtained from ATCC (American Type Culture Collection) and maintained in minimal essential medium (MEM; Gibco, Life Technologies, Grand Island, NY, USA; Ordering No: 31095_029) supplemented with 10 % fetal bovine serum (Sigma Aldrich, St. Louis, USA, cat.No.: F2442), 1 mmole/L sodium pyruvate (Sigma Aldrich; St. Louis, USA, cat.No.: S8636) and penicillin/streptomycin (100 U.mL−1/100 ng.mL−1; Sigma Aldrich; St. Louis, USA, cat.No.: P0781) in 5 % CO2 atmosphere at 37 °C.
N-terminal (N-T) fusions of GIRK1a, GIRK1d and GIRK4 with enhanced yellow fluorescence protein (eYFP) and enhanced cyan fluorescence protein (eCFP) were expressed in MCF-7 cells using the pEYFP-C1 and pECFP-C1 based constructs described in detail in . C-terminal (C-T) fusions of GIRK1a and GIRK1c with eYFP were produced by cloning the corresponding coding DNA sequence (CDS) into the plasmid pEYFP-N1 (Clontech Laboratories, Inc., Mountain View, CA, USA) using XhoI and EcoRI restriction sites. For fluorescence labelling of subcellular compartments plasmids encoding glycosylphosphatidyinositol/eCFP (GPIeCFP; for lipid rafts within plasma membrane ) and signal recognition particle receptor ß-subunit/eCFP (SrßeCFP; for endoplasmic reticulum (ER) ) were used. A vector for mammalian overexpression of fluorescence labelled G-protein β/γ subunits was generated by cloning Gγ2 CDS (Genbank Acc.No.: M37183) into the multiple cloning site (MCS) B of the pIRES vector (Clontech Laboratories, Inc., Mountain View, CA, USA) via XbaI and SalI restriction sites. Subsequently, the CDS of a fusion protein of eYFP with Gβ1 (Genbank Acc.No.: M313236; N-terminal with respect to Gβ1) was inserted into MSC B via NheI and EcoRI restriction sites. Integrity of the construct was verified by sequencing. Biological activity of fluorescence labelled G-protein β/γ subunits was verified by coexpression of the corresponding synthetic mRNAs in Xenopus laevis oocytes and subsequent electrophysiological testing for their ability to activate coexpressed GIRK ion channels composed of the GIRK1a and GIRK4 subunits (data not shown).
MCF-7 cells were transfected with the different constructs using TransFast reagent (Promega, Madison, USA, Cat. No.: E2341) and studied approx. 24 h after transfection. For stable transfection, pEYFP-C1 and pEYFP-N1 based constructs were linearized with AseI and pIRES construct with SalI, respectively, prior to transfection. Selection was started by adding 3 mg/mL G418 (Gibco, life technologies, Grand Island, USA, Ordering No.: 11811031) to the medium 24 h after the transfection. Single cell sorting was done two weeks after G418 addition. Individual clones were chosen by visual inspection using confocal Laser Scan Microscopy (cLSM). See Additional file 1: Table S1 for list of clonal cell lines that were used for the present study.
Confocal laser scan microscopy
Fluorescence images of transfected MCF-7 cells were obtained in-vivo using Leica inverted microscope with 63x H2O immersion objective (NA: 1.20) with attached laser-scan module (DMIRE2 and TCS SL2; Leica Microsystems, Heidelberg, Germany) as described previously .
RNA isolation and cDNA synthesis were performed as described previously . qPCR has been described in . Primer sequences were as follows: GIRK1a_f: 5′-GTGGAAACAACTGGGATGAC-3′; GIRK1a_r: 5′-GTTGCATGGAACTGGGAGTA-3′; GIRK1c_f: 5′- CAAGCTGCTCAAATCTCGGC-3′; GIRK1c_r: 5′-AGTTGATCTGCCCCTGTACT-3′; GIRK1d_f: 5′-CAAGCTGCTCAAAGGATGAC-3′; GIRK1d_r: 5′-GTTGCATGGAACTGGGAGTA-3′; GAPDH_f: 5′-ATGGGGAAGGTGAAGGTCG-3′; GAPDH_r: 5′-GGGGTCATTGATGGCAACAATA-3′.
Cells were fixed in NBF, embedded in agarose gel (7 %) and then processed for paraffin embedding. Target retrieval solution (pH: 9.0; Dako, Glostrup, Denmark; Product No: S236884) heated for 40 min at 150 W in a microwave was used for antigen retrieval. Slides were then washed in washing buffer (Dako, Glostrup, Denmark; Product No: S3006) and incubated with a monoclonal antibody against GIRK1 (Abcam, Cambridge, UK; cat.No: 119246; 1:50; clone 3E11). For visualization, the EnVision + dual link reagent (rabbit/mouse horse radish peroxidase, Glostrup, Dako, Denmark; Product No: K406311) was used according to manufacturer’s protocols. Immunohistochemical staining was developed by incubation of sections with diaminobenzidine (DAB; Glostrup, Dako; Product No: K406511) as a chromogenic substrate. Slides were then washed in Dako wash buffer, counterstained with Meyer’s hematoxylin (from the pharmacy of the Medical University of Graz), rinsed in tap water, dehydrated and mounted with Entellan® (Merck, Darmstadt, Germany). Sections incubated without primary antibody served as negative controls.
Analysis of vital parameters of cell lines
In order to avoid possible deviations of vital parameters that might be due to the cloning procedure itself instead of differential overexpression of GIRK1 variants, assessment of these parameters was always conducted on more than one cell line overexpressing identical constructs (Additional file 1: Table S1). As no difference in vital parameters between cell lines expressing identical GIRK1 variants was observed, these data were pooled and analyzed collectively. In order to monitor eventual effects of stable eYFP overexpression alone or of the manipulation of cellular genome on the vital parameters tested, all vital assays were performed using both MCF-7WT and MCF-7eYFP as controls.
MCF-7 cells were washed with PBS and plated into each well of Corning® BioCoat™ Fibronectin 96 Well Clear Flat Bottom (Corning, NY. USA, Cat No: 354409). Non-adherent cells were removed 150 min later by washing with PBS. Adherent cells were fixed with 2 % formaldehyde, air dried and stained with 0.1 % crystal violet (Sigma Aldrich, St.Louis, USA; Cat No: C0775) in PBS. Bound dye was solubilized with 10 % acetic acid and absorbance was measured at 550 nm using a plate reader (Labsystem Mutiskam MS). Cell-free wells served as blanks.
Cells were plated in six well plates and incubated in a cell culture incubator until reaching 80 % confluency. Proliferation of the cells were assessed based on the incorporation of the thymidine analog, 5-bromo-2′-deoxyuridine (BrdU) into the newly synthesized DNA of replicating cells (during S phase). Labeling of DNA was done by adding 10 μL of BrdU solution directly to each mL of cell culture media. (APC BrdU flow kit; BD Pharmingen, San Diego, CA, USA; Cat No: 552598). The treated MCF-7 cells were then incubated for 3 h in cell culture incubator. The cells were then stained with Anti-BrdU APC. 7-AAD (7-Aminoactinomycin D) (DNA binding dye) used in order to define cell cycle (G1/G0, S, G2/M).
Corning® BioCoat™ Matrigel® Invasion Chambers (Corning, NY. USA, Cat No: 354480) were rehydrated in MEM for 2 h at 37 °C. 1.25x105 cells/mL in 2 mL MEM (without fetal bovine serum (FBS)) were seeded into the upper compartment and 2 mL MEM with 5 % FBS as chemoattractant were added to the lower compartment of Matrigel. After 24 h incubation in cell culture incubator, non-invading cells were removed from the upper surface of the membrane by scrubbing with cotton tipped swabs. To stain the invading cells, membranes beneath the insert were cut, fixed with ice-cold methanol and stained with 0.1 % crystal violet. Invading cells were counted under microscope.
Wound healing assay
Cells were plated into 24 well plates (1x105 cells/well) and incubated for 24–72 h to achieve a confluent monolayer. The cell monolayer was scratched in a straight line with a pipet tip (VWR, USA; Cat No #53508-910). Debris was removed by washing the cells once with PBS followed by adding MEM growth medium. The plate was put into the cell observer (Zeiss Axiovert 200 M). Images were taken over the course of 72 h at 1 h interval. ImageJ software was used for analysis of the resulting time lapse videos .
Cellular velocities and motility coefficients were assessed by cell observer (Axiovert200M, Zeiss, Germany) over a time period of 72 h, as described previously .
Ex ovo chorioallantoic membrane (CAM) assay
Fertilized white leghorn chicken eggs from local hatchery (Schropper GmbH, Gloggnitz, Austria) were incubated at 37.6 °C and 70 % humidity (J. Hemel Brutgeräte GmbH & Co KG, Am Buschbach, Germany). The egg shell was cracked on day 3 of chicken embryo development, the embryo was decanted to a sterile dish and further incubated as indicated before. On day 10 of cultivation cell onplants with volumes of 20 μl were applied on vascular branches of the CAM within sterile silicon rings of 5 mm diameter (1x106 cells in 10 μl PBS mixed 1:2 with Matrigel® (Corning, NY, USA, cat.No: 356237), allowing subsequent tumor growth for 3 days . The intensity of the angiogenic response was analyzed under a stereomicroscope according as described previously .
Single channel recording from MCF-7 cells in the cell attached configuration was performed exactly as described previously for PIEZO1 mechanosensitive ion channel protein, but without application of mechanical stress to the membrane .
Statistical evaluation was performed using SPSS software in the SigmaStat environment (SigmaPlot 13.0; Systat Software GmbH, Erkrath, Germany) or using “R” software (version 3.2.1; https://www.r-project.org/). Depending on variance and distribution of the dataset, the appropriate tests were performed, as specified in the legends to the figures.