Cells and culture
Human polarized hepatic HepG2 cells (70,000 cells/cm2) were grown at 37°C under a humidified 95% air and 5% CO2 atmosphere in high-glucose Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine serum. T-lymphoblasts were prepared from isolated human peripheral blood mononuclear cells (PBMCs). Nonadherent PBMCs were stimulated with 0.5% phytohemagglutinin for 48 h and maintained in a RPMI medium supplemented with 2 U/ml IL-2 as previously described . These memory T-lymphocytes were used in experiments after they had been cultured for 7–12 days.
Cell transfection and stable expression of exogenous proteins
5 μg DNA/106 cells or 100 nM siRNA/106 cells were transfected by electroporation (200 mV, 950 μF and 480 Ω; Bio-Rad). Expression analysis was performed 24–48 h post-transfection, and the siRNA effect was analyzed 72 h post-transfection. For stable expression of exogenous proteins, transfected cells were selected by treatment with 0.75 μg/ml G-418 sulfate for at least 4 weeks after transfection. Positive cell clones were selected and maintained in drug-free medium. After several passages in this medium, > 80% of cells retained expression of the exogenous protein. For CRISPR/Cas9 gene editing, the cDNA sequence was analyzed using the Breaking-Cas tool (http://bioinfogp.cnb.csic.es/tools/breakingcas), and the selected target sequences were inserted in the pSpCas9(BB)-2A-GFP plasmid, which was a gift from Feng Zhang (Massachusetts Institute of Technology, Cambridge, MA, USA) (Addgene plasmid # 48,138; http://n2t.net/addgene:48138; RRID:Addgene_48138) . GFP-positive cells were sorted after 24 h of transfection and plated. Individual clones were tested by immunofluorescence and immunoblot analyses.
Generation of anti-human PLLP polyclonal antibody
The last 17 residues from the C-terminal end of PLLP were synthesized in the peptide synthesis facility of the CBM Severo Ochoa and conjugated to KLH. Two New Zealand white rabbits were immunized with 250 μg of the KLH-peptide and 30 days later the animals were subjected to three additional boosts of 125 μg, keeping 30-day intervals between boosts. Specificity of the sera was tested by western blot of lysates from cells expressing exogenous human PLLP and cells lacking PLLP by CRISPR/CAS9-mediated editing of the gene. Immunization and boosts were carried out in Vivotecnia (Tres Cantos, Spain).
Immunofluorescence analysis by confocal microscopy
HepG2 cells were grown on coverslips, fixed in 4% paraformaldehyde (PFA) for 15 min, rinsed and treated with 10 mM glycine for 2 min to quench the aldehyde groups. Immunostaining was performed as described . Briefly, cells were then permeabilized with 0.2% Triton X-100, rinsed and blocked with 3% bovine serum albumin (BSA) in PBS for 15 min at room temperature (RT). Cells were incubated for 30 min with the primary antibodies, rinsed in PBS and incubated for 30 min with the appropriate fluorescent secondary antibodies. Actin filaments were detected with fluorophore-conjugated phalloidin (see table). Incubation with antibodies and other fluorescence reagents were always performed at 37 °C. Confocal laser scanning microscopy was carried out using a confocal Zeiss LSM710 system coupled to AxioImager M2 microscope, a confocal Zeiss LSM 800 system coupled to an AxioObserver microscope, and a confocal Nikon AR1 + system coupled to an Eclipse Ti-E microscope. For time-lapse movies of PLLP-GFP HepG2 cells a confocal Spinning disk SpiSR10 microscope. Fixed cells were imaged on glass cover slips mounted in Fluoromount. Images were captured with 1024 × 1024 µm of resolution. The basolateral and apical intensities of ICAM-1 of WT and PLLP_KO HepG2 cells were calculated from confocal images of polarized cell colonies by measuring the fluorescence intensity of basolateral and apical areas, respectively. Images were processed with Fiji software and Imaris software for 3D volume reconstruction. When specified, XZ projections, which result from the sums of the confocal images containing the structure of interest, were shown.
Basolateral ICAM-1 labeling and transcytosis
Basolaterally labeled (BL)-ICAM-1 traffic was analyzed by incubating cells with anti-ICAM-1 antibody (0.5 µg/ml) for 30 min at 4 °C. Cells were rinsed and incubated at 37 °C for the indicated times to follow the ICAM-1 basolateral-to-apical transcytotic transport. After fixation and permeabilization, antibody-labeled receptor distribution was detected by immunofluorescence with an appropriate fluorescent secondary antibody. To identify regions of ICAM-1 internalization, a BL-ICAM-1 transcytosis assay was performed for 90 min and, before fixation, cells were incubated at 4 °C in the presence of a FITC-conjugated secondary antibody. This first secondary antibody stained the surface BL-ICAM-1 population that had not yet been internalized. Cells were rinsed, fixed and permeabilized. An incubation with a second secondary antibody conjugated to a different fluorophore was carried out to identify the BL-ICAM-1 population that could not be accessed by the FITC-conjugated secondary antibody during the first incubation. This population mostly corresponded to the internalized and transcytosed fractions of BL-ICAM-1 receptor, although a minor population of surface receptor that was not bound to the first secondary antibody, was also stained in this second incubation. Time–lapse confocal microscopy of the BL-ICAM-1 transcytosis assay was performed in HepG2 cells stably expressing GFP-ZO-1 or PLLP-GFP. Briefly, cells were seeded onto eight-well culture chambers (Ibidi) and cultured for 3 days. Antibody basolateral labeling of ICAM-1 was performed at 4 °C, cells were rinsed and incubated at 4 °C for 30 min with a TexasRed-conjugated F (ab) antibody. Cells were rinsed and placed in the humidified chamber (5% CO2 at 37 °C) of a Nikon AR1 confocal microscope. Time-lapse was performed using a 63×/1.2 water objective lens; cells were imaged in phenol-red-free DMEM medium, buffered with 10 mM HEPES, pH 7.4, at 5–10-min intervals, depending on the experiment, for the indicated times. Time-lapse acquisitions were processed using the Fiji image processing software.
T-lymphocyte adhesion assays
To measure the ability of HepG2 cells to adhere to T cells, HepG2 cells were plated onto 24-well plates (50×103/well) for 48 h. T-lymphoblasts were labeled with 0.5 μM calcein-AM for 30 min, and extensively rinsed with medium. HepG2 cells were exposed or not to 100 nM PMA for 2 h to induce depolarization and then extensively washed before performing the adhesion assay . HepG2 cells were co-cultured with T-lymphocytes in a 2:1 ratio for 15 min. After washing, cells were fixed, immunofluorescence was performed and the percentage of calcein-labeled T-lymphocytes or CD3-stained T cells adhered to HepG2 cells was measured using a fluorescence microscope.
Tissue immunofluorescence and immunohistochemistry
The livers of the mice were removed and fixed overnight in 10% neutral buffered formalin (Sigma-Aldrich) at RT. After fixation, tissues were incubated in 30% sucrose overnight and then frozen in Tissue-Tek O.C.T. compound. The sections were allowed to cool at RT and then incubated in blocking buffer (1% DMSO, 2% BSA and 0.3% Triton X-100 in PBS) for 2 h at RT. Primary antibodies were diluted in blocking buffer and incubated overnight at 4 °C. Secondary antibodies were diluted in PBS containing 0.05% BSA and incubated for 2 h at RT. Quantification of the pericanalicular vs non-pericanalicular distribution of PLLP was performed by establishing in Fiji a ROI comprising the perimeter of the BC and then widening such perimeter by 3 μm. The PLLP staining intensity in this enlarged ROI was considered to be pericanalicular and was subtracted from the intensity in the whole cellular area, which resulted in the values of non-pericanalicular intensity.
The immunohistochemical analysis of human hepatic tissue was approved by the Hospital Ethics Committee of the Hospital Universitario de Salamanca. Biopsies from healthy donors were analysed. Formalin-fixed, paraffin-embedded sections of 4 µm thickness were deparaffinized in xylene and rehydrated through a decreasing graded ethanol solution series. After suppression of endogenous peroxidase activity (3% hydrogen peroxide, 10 min) and antigen retrieval (boiling in 10 mM citrate buffer, pH 6.0), immunostaining was performed with the appropriate primary antibody. Immunohistochemical techniques were performed with an automated Leica Bond III® system. The stained protein was visualized using DAB solution (Dako), and lightly counterstained with Mayers–haematoxylin (Leica®). To ascertain the specificity of the antibody immunoreactivity, a negative control was carried out in the absence of the primary antibody. In this case, no immunolabeling was detected.
Immunolocalization of BL-ICAM-1 and PLLP-GFP by transmission electron microscopy. For transmission electron microscopy analysis, HepG2 cells were grown on 24-well plates for 48–72 h and basolateral labeling of ICAM-1 was performed as described. After 90 min of temperature shift at 37 °C, cells were fixed in 4% PFA and 0.05% glutaraldehyde (GLA) in 0.1 M PHEM buffer, pH 6.9, for 2 h at RT and 16 h at 4 °C. After extensive washing, cells samples were embedded in 10% gelatin. Sample blocks (< 1 mm3) were cryoprotected with 2.3 M sucrose at 4 °C for 16 h and rapidly frozen by immersion in liquid nitrogen. Samples were sectioned on an EM FCS cryo-ultramicrotome (Ultracut UCT, Leica) at −120 °C and collected in a mixture of 2.3 M sucrose and 2% methylcellulose solution (vol/vol 1:1). Immunogold labeling was essentially performed as previously described . For labeling of BL- ICAM-1 at 0 and 90-min time points, thawed 90-nm-thick cryosections were incubated for 30 min at RT with rabbit anti-mouse antibody (Dako, Denmark) followed by protein A conjugated to 15 nm gold (Cell Microscopy Core (CMC), Utrecht, The Netherlands) for 30 min at RT. Double labeling of PLLP and BL-ICAM-1 was performed sequentially on thawed cryosections of PLLP-GFP HepG2 cells. In brief, sections were first labeled for ICAM-1 as above and fixed with 1% GLA for 5 min to stabilize the first immune reaction. After aldehyde quenching and protein blocking, sections were incubated for 30 min at RT with a rabbit anti-GFP antibody (A-6455, Invitrogen) followed by protein A conjugated to 10 nm gold particles (CMC) for 30 min. As negative controls, sections were incubated as indicated above but omitting either the bridge antibody or the anti-GFP antibody. After labeling, sections were stained with a mix of 1.8% methylcellulose and 0.4% uranyl acetate and visualized at 80 kV with a JEM 1010 (JEOL, Japan) electron microscope equipped with a F416 CMOS 4 K camera (TVIPS, Germany) or a JEOL JEM-1400 Flash electron microscope equipped with a One View CMOS 4 K camera (Gatan, United States).
Protein extraction and western blot
Cell lysates were prepared using Laemmli buffer supplemented with a cocktail of protease inhibitors. The lysates were heated at 95 °C for 5 min and cleared by centrifugation at 14,000 rpm. for 5 min. The samples were loaded on acrylamide gels and transferred onto an Immobilon-PVDF membrane (Millipore), which was blocked in 5% milk and incubated overnight with the indicated primary antibody. Anti-rabbit or anti-mouse horseradish–peroxidase-(HRP)-conjugated secondary antibodies were then used and the antibody–protein complexes were visualized using ECL (GE-Healthcare). The bands intensities were quantified using Fiji software. For tissue extraction, mice were euthanized and 100 mg of liver from each animal were lysed in ice-cold 20 mM Tris–HCl pH 7.5, 5 mM EDTA, 0.2 mM EGTA using a polytron tissue homogenizer. Cell debris was removed by centrifugation for 5 min at 2000× g at 4 °C and supernatant protein concentration was measured with BCA protein assay (Thermo Fisher Scientific) and subjected to western blot analysis with the indicated antibodies.
Biotinylation of ICAM-1-proximal proteins: BioID assay
To generate an expression plasmid containing the construct ICAM-1-BirA*, the sequence coding for GFP in the ICAM-1-GFP was substituted by the BirA* sequence  obtained from the Cav1-BirA* plasmid, kindly provided by Prof. I. Correas (Centro de Biología Molecular Severo Ochoa), with BsrGI and AgeI enzymes (New England Biolabs). The expression vector coding for ICAM-1-BirA* was transfected into HepG2 cells by electroporation and cell clones stably expressing ICAM-1-BirA* were selected with G-418 as previously described . ICAM-1-BirA* HepG2 cells were cultured on 10 cm diameter plates and after 48 h, incubated with 50 μM biotin for 16 h, lysed and subjected to a pull–down assay of biotinylated proteins with neutravidin-agarose (Thermo Scientific) as previously described . Lysates and pull–down pellets were analyzed by western blot.
PLLP-GFP HepG2 cells were grown for 72 h and washed once in cold PBS and lysed in 400 μl of TNE buffer (50 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA) containing 1% Triton-X100 and protease inhibitor cocktail. Lysates were incubated with 20 μl protein A-coated Sepharose (Sigma-Aldrich) previously conjugated with 5 μl of anti-GFP antibody by overnight incubation, for 3 h at 4 °C with agitation; rabbit IgG antibody was used as a control. Antibody-conjugated beads were rinsed in TNE + TX100 buffer five times and dried by aspiration. Immunoprecipitated proteins were eluted in 20 μl of Laemmli´s buffer and analyzed by western blot. For immunoprecipitation assays of the BL-ICAM-1 population, cells were basolaterally labeled with anti-ICAM-1 antibody as described, then incubated at 37 °C for the indicated times, and immediately lysed in TNE + TX100 buffer. The post-nuclear supernatant of the lysates was incubated with protein G-Sepharose for 3 h at 4 °C with agitation, rinsed and analyzed by western blot.
Generation of HepG2 tumors
Athymic nude mice were purchased from Charles River and kept in standard conditions of 22 ± 2 °C temperature, 45–55% humidity, rate of 12/12 h light/dark cycle, and food and water ad libitum in the CBM Severo Ochoa animal facility. Before implantation, HepG2 cells were trypsinized, washed gently in PBS, and resuspended in Optimem. 1 × 106/100 μl of HepG2 control or PLLP_KO cells were implanted subcutaneously on the left hind flank of each mouse. Tumor growth was monitored every 7 days by palpation until the tumor was measurable with calipers, whereupon the monitoring frequency increased to once every 2 days. Tumor size was calculated as its volume (mm3) using the formula [[length (mm) × width (mm)] × 0.5236] x width (mm). For ethical reasons, mice were sacrificed when tumor volume reached 1,500 mm3. Tumors were harvested, washed in PBS, fixed in 4% PFA for 24 h at 4 °C and processed for tissue immunofluorescence as described. All these animal experimentation procedures conformed to the European Guidelines for the Care and Use of Laboratory Animals (Directive 86/609) and were approved by the Ethical Committees for Animal Experimentation of Universidad Autónoma de Madrid and the Comunidad Autónoma de Madrid, Spain.
Quantification and statistical analysis
Data are expressed as the mean plus standard deviation (SD) or mean plus the standard error of the mean (SEM). Student’s two-tailed t tests or two-way ANOVAs were used to establish the statistical significance (p < 0.05) of group differences, depending on the experiment. In all cases, data from at least three independent experiments were used. All calculations were performed using Prism 7 software.