rhIns was kindly provided by Laboratorios Beta (Buenos Aires, Argentina). IGF-II-biot was from IBT system (Reutlingen, Germany) and human recombinant IGF-II was from Gro Pep (Adelaide, Australia). BAC-Ins was from Sigma (Munich, Germany). Polyclonal rabbit anti-ERK2, polyclonal rabbit anti-CD63 (Lamp 1) and polyclonal rabbit anti-IR-α-subunit (N20 and H78) were from Santa Cruz Biotechnology (Paso Robles, CA). EEA1 marker (rabbit) was from Abcam (Cambridge, MA). Monoclonal mouse anti-phosphorylated-tyrosine (PY20) was from BD Transduction Laboratories (Franklin Lakes, NJ). Monoclonal rabbit anti-phosphorylated-IR-β subunit (Tyrosine 1361), monoclonal mouse anti-IR-β subunit and polyclonal rabbit anti-phosphorylated (p42/44) MAPK were from Cell Signaling Technology (Beverly, MA). The monoclonal rabbit anti-phosphorylated-IR-β subunit (Tyrosine 1361) from Cell Signaling (cat # 3023) only detects transfected levels of IR β subunit and slightly cross-reacts with activated IGF-I receptor. The streptavidin conjugated QD655 and secondary antibodies conjugated with Alexa fluor 555 were from Molecular Probes, Invitrogen (Eugene, OR). Streptavidin-atto 550 was from ATTO-TEC Gmbh (Siegen, Germany). Lipofectamine Reagent 2000, DNase I and Trizol were from Invitrogen (Carlsbad, CA). High Capacity cDNA Reverse Transcription kit was from Applied Biosystem (Foster City, CA). Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation Assay (MTS) and Gotaq DNA polymerase were from Promega (Madison, WI). The plasmid containing the luciferase reporter gene downstream of seven binding sites for AP-1 (pAP-1-Luc) was provided by Dr. Omar Coso (IFIBYNE, Argentina).
pcDNA3-IR-B: cDNA of IR-B was provided by Dr. Axel Ullrich (Max Planck Institute for Biophysical Chemistry, Germany) and amplified by PCR using primers containing HindIII restriction site (IR-Forward: 5′-aagcttatggccaccgggggccgg-3′) and NheI and XbaI sites (IR-Reverse: 5′-tctagagctagcgaaggattggaccgaggcaaggtc-3′). The product was digested with HindIII and XbaI enzymes and cloned into the pcDNA3 vector. pcDNA3-IR-B-GFP, pcDNA3-IR-B-SCFP and pcDNA3-IR-B-SYFP: peGFP-C3 (Clontech), pSCFP-3AC or pSYFP-2F (provided by T. W. Gadella Jr., University of Amsterdam, The Netherlands) were digested with NheI and ApaI and cloned into pcDNA3-IR-B.
Cell culture and transfections
Cell culture reagents (Dulbecco’s modified Eagle’s medium (DMEM) Glutamax, Optimem, fetal bovine serum (FBS), trypsin and antibiotics) were from GIBCO (Grand Island, NE). HeLa cells were maintained in DMEM supplemented with penicillin, streptomycin and 10% FBS at 37°C in 5% CO2. Human cervical carcinoma HeLa cells (ATCC-CCL-2) were plated at 1×105 cells/well in 24 well plates and onto 12 mm glass coverslips (for microscopy experiments) or at 2.5×105 cells/well in 12 wells plates (for Western blot experiments) one day before transfection in DMEM supplemented with 10% FBS without antibiotics. Cells were transfected with Lipofectamine Reagent 2000 for 5 hours using 0.4 μg DNA and 1 μl Lipofectamine per well for 24 well plates and 0.8 μg DNA and 2 μl Lipofectamine per well for 12 well plates. After transfection cells were cultured for further expression in complete medium.
Following stimulation with rhIns, BAC-Ins, IGF-II or IGF-II-biot cells were lysed in buffer containing 100 mM Tris–HCl pH 6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol, 200 mM β-mercaptoethanol, vortexed for 20 sec and heated 5 min at 100°C. After 10% SDS-PAGE and transfer, membranes were blocked in 5% w/v non-fat dried milk in 0.1% Tween-TBS buffer (TTBS) for 1 hour, washed and incubated overnight at 4°C with primary antibodies diluted in 5% bovine serum albumin (BSA)/TTBS (anti-IR-β subunit: 1/500, anti-ERK2: 0.2 μg/ml, anti-phosphorylated-p44/42 MAPK: 1/500) or in 2% BSA/TTBS (anti-phosphorylated-Tyrosine: 2 μg/ml). Membranes were incubated with secondary antibodies for 1 hour and washed with TTBS. The blots were developed by chemiluminescence with a Bio-Imaging Analyzer Bas-1800II and Image Gauge 3.12, FUJIFILM.
Luciferase reporter assay
Cells seeded onto 24 well plates (1 × 105 cells/well) the day before were transfected using 0.3 μg pcDNA3-IR or pcDNA3 (empty vector, EV) and 0.05 μg pAP1-Luc. After 24 hours, cells were starved one day, and then stimulated for 16 hours with 1.6 nM EGF, 100 nM rhIns, 100 nM IGF-II, 1.6 nM EGF + 100nM rhIns or 100 nM IGF-II + 100 nM rhIns. Luciferase activity was determined using Luciferase Reactive and Reporter Lysis Buffer from Promega (Madison, WI) and normalization to the control (non-stimulated cells) was performed (fold induction). The results were expressed as the mean of at least three independent experiments ± s.e.m. The p values were estimated using Student’s T test (2 tails).
Cell proliferation experiments
Cells were transfected with pcDNA3-IR or the EV, 24 hours after transfection they were treated with 1% FBS overnight before stimulation with 0.1 nM, 1 nM or 10 nM rhIns or IGF-II for 2 days. Cell proliferation was measured using a non-radioactive assay (MTS) according to manufacturer procedures. The results were normalized to those obtained for the cells incubated in 1% FBS over all the experiments. The results were expressed as the mean of at least three independent experiments ± s.e.m. The p values were estimated using Student’s T test (2 tails).
Labeling in vivo with QDs-IGF-II-biot or BAC-Ins and internalization
Before the experiment cells expressing IR-B, IR-B-SYFP or IR-B-GFP were starved overnight, washed with Tyrode’s buffer (135 mM NaCl, 10 mM KCl, 10 mM MgCl2, 1mM CaCl2, 10 mM HEPES pH 7.2, 0.1% BSA) at room temperature or 15°C (as indicated in figure legends) and incubated with 50 nM BAC-Ins or IGF-II-biot for 15 min, washed with Tyrode’s and incubated with 0.5, 1.0, 2.0 or 4.0 nM QD655 for 10 min in darkness, washed and either fixed in 3.7% paraformaldehyde (PFA) on ice for 20 min or incubated at 37°C in DMEM for different periods of time before fixation. When acid treatment was applied, cells were incubated for 5 min (microscopy) or 2 min (flow cytometry) at room temperature with, 0.5 M NaCl, 0.1 M Na-glycine pH 3.0 and then fixed in 3.7% PFA for microscopy experiments or directly analyzed by flow cytometry.
After overnight starvation, transfected cells were stimulated with 100 nM rhIns, IGF-II or IGF-II-biot for 5 min, washed with cold PBS and immediately fixed in cold methanol for 30 min at -20°C, blocked with PBS/0.3% Triton X-100/1% BSA for 1 hour at 37°C and incubated with anti-phosphorylated-IR-β subunit (0.3 μg/ml) overnight at 4°C. The following day the samples were incubated with a secondary antibody conjugated with Alexa fluor 555 for 1 hour at 37°C and washed. IR activation was monitored by confocal microscopy. To study the expression of IR-B-GFP we followed the same strategy but we did not starve the cells before the assay. We used anti-IR-α-subunit (N20 and H78) (0.2 μg/ml), the following day we incubated the cells with a secondary antibody conjugated with Cy3. For endocytosis immunofluorescence, HeLa cells expressing IR-B were labeled with BAC-Ins and QD655, induced internalization and fixed in 3.7% PFA. Immunofluorescences were performed with 4 μg/ml anti-CD63 or 1.5 μg/ml EEA1.
Confocal laser scanning microscopy was performed with: (i) an Olympus Fluoview FV 1000 microscope with a UPLSAPO 60× 1.2 NA water immersion objective. Excitation and emission filters were as follows: excitation SCFP, 405 nm; emission SCFP, band pass (BP): 430–470 nm; excitation GFP, 488 nm; emission GFP, BP: 505–605 nm; excitation SYFP, 515 nm; emission SYFP, BP: 535–565 nm; excitation DAPI, 405 nm; emission DAPI, BP: 430–470 nm; excitation QD655, 405 nm; emission QD655, BP: 655–755 nm; excitation Alexa fluor 555, 543 nm; emission Alexa fluor 555, BP: 560–620 nm; (ii) a Zeiss LSM 510 Meta microscope with a Plan-Apochromat 63× 1.4 NA oil immersion objective. Excitation and emission filters were as follows: excitation FITC, 488 nm; emission FITC, 510–563 nm.
We always used the sequential mode for image acquisition.
In the experiments where imaging was performed with a Zeiss LSM 510 Meta microscope we used a C-Apochromat 6× 1.2 NA water immersion objective. Excitation and emission filters were as follows: excitation GFP, 488 nm; emission GFP, BP: 500/20 nm; excitation QD655, 488 nm and 458 nm; emission QD655, LP: 650 nm.
Wide field microscopy was performed with a Zeiss Axiovert S100 with a 63× 1.25 NA oil immersion objective (Zeiss), a mercury arc lamp excitation and filters suitable for GFP, Cy3 and DAPI signals. Camera: Hamamatsu Orca CCD C4742-95.
Confocal microscope images were processed with Matlab (TU Delft, The Netherlands) and Image J (National Institutes of Health). The background of each channel (mean of empty region) was subtracted and in some cases a median filter was applied (radius: 1 pixel) only for presentation. No filter was applied in quantitative analyses. The z-stacks (64 frames, 0.16 μm step size) were processed by deconvolution using Scientific Volume Imaging Huygens Professional Version 3.6 software, applying a Quick Maximum Likelihood Estimation (QMLE) algorithm.
For quantitative internalization experiments, we defined time “0 min” as the end of labeling-washes, and t = 10, 20 and 150 minutes of incubation at 37°C.
Segmentation (membrane and interior)
Channel backgrounds (median) were subtracted. Segmentation was performed for each cell using the SYFP signal. After cell segmentation the pre-membrane was defined as the difference image of the cell and a binary erosion (iterations: 5–20; alternating connectivity); we evaluated the results visually. The pre-interior was defined as the difference between the cell and the pre-membrane. A QD
marked red pixels. With this mask a membrane was defined as the product of the QD
and pre-membrane, and interior as the product of the QD
and pre-interior obtained previously.
Estimation of the relative amount of internalization
Values in membrane and interior were summed for red and green channel, and also sizes were measured. To compute the relative amount of internalized red fluorescence we estimated QD
as the sum of QD
and we calculated for each cell the ratio QD
. This “internal calibration” approach was chosen to remove the influence of the amplifier gain and the zoom factor for each image acquisition condition. The expression levels were estimated as the mean of the SYFP signal (sum of SYFP/cell size) and for the statistical analysis only cells with similar level of IR-SYFP expression were considered.
Manders coefficients and PDM graphs were performed with the Image J plugin Intensity correlation analysis. The PDM graphs show the contribution of each pixel to the colocalization coefficient.
Flow cytometry was performed with a Becton Dickinson FACSAriaII. The filters, lasers and dichroic mirrors (DM) were as follow: (i) SYFP: excitation 488 nm, DM LP: 502 nm, emission BP: 530/30 nm; (ii) QD655: excitation 488 nm, DM LP: 655 nm, emission BP: 660/20 nm. Data analysis was performed with WinMDI Version 2.9 software. Positive events for SYFP signal were selected taking into account basal auto-fluorescence of control cells (non-transfected cells). QD655 histograms were performed with the population of events SYFP positive (transfected cells) and overlays of histograms from cells incubated at 37°C for 20 or 90 min and non-incubated cells were displayed. A geometric mean was calculated for each histogram using the WinMDI. In each histogram a marker M1 was determined including approximately 4% of the events at the initial moment (0 min). For each internalization period the percentage of events inside region M1 was measured and the normalization was done with respect to the initial step (0 min) (Marker/0 min).
The data for internalization experiments (microscopy and flow cytometry) were fitted by Origin 8.6 using sigmoideal logistic curve.
The results were expressed as the mean ± s.e.m. p values were estimated using two-tailed Student’s t-tests.
For supplementary methods see Additional file 7.