Cell Culture and treatments
Mouse hepatic progenitor MLP29 and mouse HCC Hep16 cells were maintained in RPMI 1640 medium supplemented with 10% Fetal Bovine Serum (FBS) at 37°C in a humidified 5% CO2 atmosphere. For experiments, after cell plating, the old medium was replaced with fresh medium containing low FBS (0.1%) and the cells were maintained under this serum starvation condition for at least 16 hours. Then the cells were treated with 10 μg/ml of a specific function-blocking anti-α5β1 integrin antibody (clone BMB5) purchased from Chemicon-Millipore (Temecula, CA, USA) or 20 ng/ml of HGF/SF1. When appropriate, 20 μM of U0126 (ERK inhibitor) was used for 24 hours. Control experiments were carried out by adding equal amounts of DMSO, ethanol or an IgG control Ab.
Antibodies and reagents
MEK1 and MEK2 specific inhibitor U0126 was purchased from Calbiochem (San Diego, CA), dissolved in DMSO, and stored in the dark as a 10 mM stock solution at -20°C. The components of the ECM, fibronectin (FN) and vitronectin (VN) were purchased from Sigma (St Louis, MO), laminin (LMN) was purchased from Chemicon (Temecula, CA) and collagen type I (COL I) was purchased from BD Biosciences-Europe (Erembodegem, Belgium). hrHGF was purchased from Peprotech (Rocky Hills, NJ). Specific antibodies against β1 and β3 integrin subunits, CK19, Acetyl histone H3 (Lys9/14), and tri-Methyl histone H3 were purchased from Chemicon-Millipore (Temecula, CA), and E-Cadherin antibody was from R&D systems. Phospho p42/p44 MAPKinase antibody (Thr 202/Tyr204), total MAPKinase; phospho PKB/Akt (Ser 473) and total PKB/Akt antibodies were purchased from Cell Signaling Biotechnologies (Beverly, MA, USA). β-actin, Ep-CAM and AFP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Pan-centromeric STAR-FISH™ probe were purchased from Cambio (Cambridge, UK).
Sub-cellular localization of β1 integrins, Ac3K9/k14H3, Me3K9H3 and phospho-ERKs was assessed by immunofluorescence. Briefly, the cells were fixed in 4% paraformaldehyde (w/v) in PBS, rinsed in TBS and then permeabilized in 0.5% TBS-Triton X-100 (T-TBS 0.5%) for 10 minutes at RT. After blocking with 2% BSA in TBS for 10 min at RT, the cells were incubated with the primary antibody for 1 hour at RT. The detection was performed with secondary antibodies conjugated to FITC or Texas Red. To study the actin cytoskeleton and morphological changes, the cells were stained with phalloidin conjugated to Texas-red. After exhaustive washings with cold TBS, the slides were mounted with Vectashield containing DAPI (Vector Laboratories, CA, USA). Images were acquired with an Olympus BX61 epifluorescence microscope or a Leica DMIRE-2 confocal microscope.
Flow cytometric evaluation of RGD-binding integrins
MLP29 hepatic progenitor and Hep16 HCC cells were seeded and allowed to attach for 24 hours. Cells were then harvested by scraping, subsequently counted, and then incubated with antibodies against the α5β1 heterodimer and β1 integrin subunit (3 μg/500 × 103 cells/100 μl) for 1 hour at 4°C. After that, cells were washed twice with cold PBS containing 1% FBS, and incubated with a CY2-conjugated secondary antibody for 30 minutes at 4°C. Cells were then analyzed in a FACScanto II flow cytometer (Becton Dickinson). FACS Diva software (Becton Dickinson) was used for data acquisition and analysis.
Flow cytometric analysis of Ac3K9/K14H3 and DNA content
MLP29 hepatic progenitor cells were treated with the α5β1 blocking antibody or HGF/SF1 for 6 hours, and then the cells were harvested. For Ac3K9/K14H3 detection, cells were fixed in 1% paraformaldehyde for 15 minutes and stored in 70% ethanol at -20°C. After fixation, cells were permeabilized with PBS-0.25% Triton X-100 for 10 minutes, washed with cold PBS-1% FBS and incubated with an anti-Ac3K9/K14H3 antibody for 2 hours at room temperature. Then, the samples were washed twice and incubated with a FITC-conjugated secondary antibody. For DNA staining, the cells were washed again and treated with RNAse A (0.2 mg/ml) in PBS at 37°C for 20 minutes. Propidium Iodide (20 μg/ml) was added to the cell suspension, and incubated 30 minutes at RT protected from light. FACSDiva software (Becton Dickinson) was used for data acquisition and analysis.
Cell adhesion studies
Cell adhesion was assessed by the MTT assay using the CellTiter 96® Non-Radioactive Cell Proliferation assay (Promega Corporation, WI). Briefly, MLP29 and Hep16 cells were seeded at 50 × 103 cells/well in 96-well plates coated with FN (10 μg/ml), VN (1 μg/ml), LMN (10 μg/ml), COL I (0.05 mg/ml) and BSA (0.2 mg/ml) as a control, and allowed to attach for 1 hour. After extensive washings, the medium was aspirated carefully and the dye solution added. After 3-5 hours of incubation, the reaction was stopped with the "Solubilization/Stop" buffer provided with the kit. Following overnight incubation at 37°C, absorbance at 570 nm was determined with a SpectraMax M2 plate reader (Molecular Devices, Sunnyvale, CA, USA).
Cell migration and cell invasion assays
To study cell migration we performed the wound healing assay. MLP29 and Hep16 cells were plated seeded on six wells plastic tissue culture dishes and grown to confluence. Then, the cell monolayer was scratched with a p200 pipette tip, washed twice, and incubated in growth medium with 0.1% FBS containing 10 μg/ml of α5β1 functional-blocking antibody or 40 ng/ml of HGF/SF1 for 20 hours. Microscope images were taken from at least three fields for each experiment. The distance covered by the migrating cells was calculated as the mean of six different measurements along the scratch using the Gimp 2.0 image software.
To assess cell invasion, we used the QCM™ 24 wells Cell Invasion Assay from Chemicon-Millipore (Temecula, CA, USA) according to manufacturer's instructions. Briefly, 500 × 103 MLP29 or Hep16 serum starved cells were loaded into an insert containing an eight μm pore size and polycarbonate membrane coated with ECMatrix™ in the presence of α5β1 functional-blocking antibody or HGF/SF1 at the concentrations stated above and incubated for 48 and 72 hours. Invaded cells at the bottom of the membrane were detached, lysed and detected by the CyQUANT™ GR Dye in a SpectraMax M2 plate reader (Molecular Devices, Sunnyvale, CA, USA).
Immunoblotting analysis of ERK1/ERK2 MAPK, p-ERK1-ERK2 MAPK, AKT, pAKT, integrin sub-units and histone acetylation
Cells were cultured in a serum free medium for 16-18 hours and then stimulated with HGF/SF1 or treated with α5β1 functional-blocking antibodies for up to 24 hours. After treatment, cells were harvested, washed twice in PBS, and lysed with the Cell Lysis Buffer (Cell Signaling Biotechnologies) and 1 mM phenylmethylsulfonylfluoride for 30 minutes on ice. The lysates were then cleared by centrifugation (14000 rpm for 15 minutes at 4°C). Protein concentration was determined using the Pierce protein assay kit (Rockford, IL, USA). For analysis of ERK1/2 MAPKs and Akt, equal amounts of protein (20 μg) were resuspended in 5× Laemli buffer (10 minutes at 100°C), resolved by electrophoresis on a 10% SDS-PAGE, and transferred onto nitrocellulose membranes (Amersham Biosciences). For analysis of histone H3 acetylation, 50 μg of protein was resolved on a 15% SDS-PAGE and transferred onto a nitrocellulose membrane. The membranes were then blocked with TBS-T (25 mM Tris-HCl, 150 mM NaCl (pH 7.5), and 0.05% Tween 20) containing 5% (w/v) non-fat dry milk and incubated overnight at 4°C with specific antibodies. After incubation, the membranes were washed in TBS-T, and HRP-conjugated anti-rabbit, anti-mouse or anti-goat antibodies were added for 1 hour as secondary antibodies. Immunodetection was performed using the Western Lighting chemiluminescence reagent from Perkin Elmer (Boston, MA, USA).
Fluorescent in Situ Hybridization (FISH)
Cells were fixed following protocols designed for the preservation of the three dimensional structure of the nuclei. For centromere analysis, the cells were fixed in 4% paraformaldehyde in PBS 0.3× (w/v) for 10 minutes and then permeabilized in PBS/Triton X-100 0.5% for 20 minutes at RT and transferred to 20% Glycerol/PBS. After 1 hour, the cells were further permeabilized by freezing and thawing cycles in liquid nitrogen. The probe was dissolved in 50%formamide/20% dextran-sulfate, denatured and incubated along with the denatured cell-targets in a humid chamber at 37°C overnight. For locus-specific 3D FISH, the fixation of the cells was performed according to the protocol described previously , with slight modifications. Briefly, the coverslips were fixed for 10 minutes in 4% paraformaldehyde in PBS (w/v). After permeabilization in 0.5% saponin (w/v) PBS/Triton X-100 0.5% (v/v) for 30 minutes, the slides were washed in PBS for 2 minutes at RT and treated with 0.1 N HCl for 20 minutes. Next, the cells were washed with PBS and 2 × SSC for 2 minutes, treated with RNAse A, transferred to 2 × SSC/50% formamide 0.1% NaAz and stored at 4°C. We used probes prepared with the BAC clones RP23-303A11 y RP23-343N12 specific for the Itgb1 or the Itgb3 genes, respectively. The probes were labeled by nick translation with dUTP conjugated with biotin (Roche) or digoxigenin (Roche). Before hybridization, the probes were pre-denatured at 80°C for 5 minutes, then denatured along with the target DNA at 75°C for 5 minutes and incubated at 37°C for 72 hours in a humified chamber. For the post-hybridization washings, the coverslips were immersed in 50% formamide/2 × SSC, 1 × SSC, and 4 × SSC/tween-20 0.1% (v/v) (4T) each for 5 minutes at 47°C. Following one more wash with 4T at RT, the coverslips were blocked with 3% BSA in 4T (wt/v), washed in 4T at RT for 5 minutes and incubated for 1 h with antibodies against biotin or digoxigenin in blocking solution. Then the coverslips were incubated with the respective secondary antibodies for 1 hour, washed twice with PBS for 5 minutes and mounted with Vectashield containing DAPI (Vector Laboratories, CA, USA).
Image acquisition and analysis
Stacks of images scanning the whole nucleus were acquired with an axial separation of 250 nm using a laser-scanning microscope Leica DM IRE2 (Leica Microsystems Heidelberg GmbH). For quantitative analysis of chromocenters and Me3K9H3 foci, regions of interest (ROI) including each nucleus were directly generated on the z-stack using the Volocity.4.3® software (Improvision, Image, Processing and Vision Company Limited) and then the intensity level, volume and center of mass of all objects contained within each ROI was automatically recorded. Between 500-1000 elements (chromocenters or Me3K9H3 foci) per treatment were analyzed. For statistical analysis, the "proportions test" was used to compare the average number of elements per nucleus for each treatment. For the comparison of mean volumes and mean fluorescence intensity, we applied the "t-student" test. Fluorescence intensity of Ac3K9/K14H3 was directly measured on the Z-stack using the Volocity.4.3® software (Improvision, Image, Processing and Vision Company Limited).
To determine the 3D radial position of any given signal, the shortest distance from the center of mass of the nucleus to the periphery, which included the center of mass of the fluorescent signal, was directly measured on the z-stack using the Volocity.4.3® software. The absolute distances from the nucleus center to the gene were normalized as a fraction of nuclear radius, to account for natural variations in nuclear size that may influence positioning. Cell nuclei were subdivided into five concentric shells each corresponding to 20% of the nuclear radius, and the radial positioning data was binned into these five sub-domains. Graphs were made using Microsoft Excel Software. For quantitative measurements, 50-70 nuclei from multiple experiments were analyzed. Statistical differences (p < 0.05) between the distributions of a gene in different conditions were determined using the 1D Kolmogorov-Smirnov test.
Microarray data acquisition and analysis
RNA was isolated from cell plates subjected to the different treatments using the RNAeasy Mini kit (Qiagen) and treated with RNAase-free DNAase (Qiagen). The cDNA was synthesized from 1 μg of RNA using the Reverse Transcriptase kit (Promega) following the manufacturer's recommendations. Expression profiling was performed using the Affimetrix GeneChip ® technology, following the protocols recommended by the manufacturers. Affymetrix raw files (.cel, Mouse430A_2) obtained from untreated control cells, treated with anti-α5β1 antibodies, and stimulated with HGF/SF1 were collectively analyzed by using the R package "AFFYLMGUI" (http://www.bioconductor.org) . Data were background corrected by the rma method . The p-values obtained by testing for differentially expressed genes were calculated using "AFFYLMGUI" and exported as text. Extraction of GO terms for all genes on the Mouse430A_2 chips was used for functional analysis. Hypergeometric p-values for testing over representation of genes in GO categories were calculated as described in Masseroli et al., and the p-values were corrected for multiple testing by the Holm method [26, 27]. The gene set enrichment analysis was done as described previously [28, 29]. The permutation statistics for the heat-plots were calculated for each gene set. This was done by considering the expression level for all genes of each set defined by Molecular Signatures Database. The p-values for the heat-plots were calculated as ndown/N for down-regulated genes and as 1-nup/N for up-regulated genes, where ndown is the number of times a permutated data set resulted in a down regulation that was stronger or equal to the one observed for the unpermutated data. nup was calculated in a similar way for up-regulated genes. N was the total number of permutation which was set to 10000. To assess if a specific gene set was up- or down-regulated for a specific sample class the expression values from a specific class were compared with the expression values in all other sample classes . The calculation of 1-nup/N for up-regulated gene sets allows co-plotting both up and down-regulated gene sets in the same heat-diagram. Mouse genes were mapped to human genes by using HomologGene available at NCBI. Microarray data is publicly available at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE23853, Gene Expression Omnibus (GEO) accession number GSE23853.