Mouse anti-Rac1 monoclonal antibody was purchased from Thermo Scientific. Goat anti-plectin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse anti-phosphorylated FAK monoclonal antibody (anti-FAKp) was acquired from BD Transduction Laboratories™ (Franklin Lakes, New Jersey, USA). Secondary antibodies including Cy2-conjugated anti-mouse IgG and Cy3-conjugated anti-goat IgG used for immunofluorescence microscopy, horseradish peroxidase-conjugated goat and mouse antibodies used for Western blotting analysis, as well as biotin-conjugated goat and mouse antibodies used for immunohistochemistry were purchased from Jackson Immuno Research Laboratories (West Grove, PA, USA).
Chang liver cells (CCL-13, obtained from ATCC) were derived from normal human hepatic tissue. Dulbecco’s minimum essential medium (DMEM) supplemented with 10% fetal bovine serum, 50 unit/ml penicillin and streptomycin was used for culture at 37°C in presence of 5% CO2. Medium was replaced and confluent growth was split regularly.
HCC tissue specimens
Five primary HCC specimens without prior neo-adjuvant therapy were obtained from surgical resection operated in the Department of Surgery, Chang Bing Show Chwan Memorial Hospital, Changhua County, Taiwan. Pathological diagnosis confirmed that all specimens were neoplastic grade II. Paraffin-embedded blocks of the specimens were prepared as usual protocols.
Transient knockdown of plectin in Chang liver cells
To study the biological effects of plectin deficiency, transient knockdown was performed by using RNA interference techniques. Small interfering RNA (siRNA) specific for plectin transcripts was purchased from Dharmacon, Inc. (Lafayette,CO). The pre-designed siRNA kit contains a mixture composed of four fragments of siRNA targeting to the sequences of conserved regions encoding all plectin isoforms. Chang liver cells were grown on a 60mm petri dish to 60% confluence. For introducing the siRNA into cells, the siRNA was diluted with serum free medium (1:100, v/v) and then gently mixed with pre-diluted (1:50, v/v) Lipofectamine 2000 (Invitrogen, USA). The mixture was left at room temperature for 20 minutes. Afterwards the mixture was added to the petri dishes with gentle shaking. The transfected culture will be available for further assays after 48 hours.
Trans-well migration assay
Cell motility was examined by using QCM™ 24-Well Colorimetric Cell Migration Assay kit (Millipore). The siRNA-transfected Chang liver cells were harvested and then transferred into the 24-well plate inserted with trans-well. Cellular migration was determined in accordance with the manufacture’s protocol.
Preparation of p21-activated kinase (Pak)
The Pak-expressing E.coli BL21 strain was grown in 500ml ampicillin supplemented-LB medium to the density with OD600 value 0.6-0.8. Pak expression was induced by adding 500μL isopropyl β-D-1-thiogalactopyranoside (1mM) and maintains further shaking at 4°C for 3 hours. The bacterial culture was harvested by centrifugation (6000g) at 4°C for 15 minutes and the pellet was washed with 25ml washing buffer containing 62.5μL phenylmethylsulfonylfluoride (PMSF), and 250μL protease inhibitors cocktail. The washed bacteria were lysed by sonication (Misonixsonicator 3000) as protocol’s instruction. The lysate was added with 500μl Triton X-100 and incubated at 4°C for 30 minutes. The supernatant was collected after centrifugation (12000g) at 4°C for 20 minutes. The glutathione S-transferase (GST)-conjugated beads (200μl) was added to the collected solution and incubated at 4°C for 30 minutes with continuous shaking. The Pak-bound beads were further collected by centrifugation (5000rpm) at 4°C for 3 minutes and then washed with 1ml 1x phosphate buffered saline (PBS) buffer. The washing procedures were repeated for 3 times. The Pak-GST-conjugated beads can be collected and suspended with 1ml lysis buffer (25mM Tris-base, 150mM NaCl, 1% NP-40, 5% Glycerol, 1mM ethylenediaminetetraacetic acid (EDTA), 100μM sodium orthovanadate, 200μM PMSF and protease inhibitors cocktail).
Ras-related C3 botulinum toxin substrate 1 (Rac1)-GTPase pull-down and detection assay
A 60mm petri dish culture of mock and plectin knockdown Chang liver cell were separately harvested and suspended with lysis buffer (25mM Tris-base, 150mM NaCl, 1% NP-40, 5% Glycerol, 1mM ethylenediaminetetraacetic acid (EDTA), 100μM sodium orthovanadate, 200μM PMSF and protease inhibitors cocktail). Supernatant of both samples were collected by centrifugation (11000g) at 4°C for 10 minutes. The protein concentrations were determined by bicinchoninic acid assay (BCA assay) and adjusted to a final amount of 500μg in a volume of 700μl. The Pak-GST-conjugated beads (10μl) prepared as the previous description was then added to each samples and were continuously shaken at 4°C for 2 hours. The incubated samples were further washed with lysis buffer and collected by centrifugation (4500rpm) at 4°C for 5 minutes. The washing procedures were repeated for three times. The washed samples were rinsed with distilled water and were eligible for determining the Rac1-GTPase activity by Western blotting analysis.
Western blotting analysis
Total cellular lysate and Rac1-GTPase pull-down complex were separately loaded for running a 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE). Followed by the completion of electrophoresis, the gel was transferred onto a polyvinylidene difluoride (PVDF) membrane by using semi-dry transferring method (Bio-Rad). The transferred membrane was blocked with 5% non-fat milk (in phosphate buffered saline with Tween-20 (PBST) buffer) for 1 hour and then washed three times for 5 minutes with PBST prior to adding the diluted primary antibody (1:1000 in PBST). The hybridization was undergone for one hour and the hybridized membrane was washed with PBST three times for 5 minutes prior to adding the diluted secondary antibody (1:5000 in PBST). At last, the chemiluminescent reagent was added to the washed membrane and the image was developed by the Chemiluminescence Imaging System (Fuji, Japan) as manual’s instruction.
The plectin siRNA-transfected Chang liver cells were grown in a 24-well plate (4 × 103 cells per well) for 48 hours. Cells were washed with ice-cooled PBS (137mM NaCl, 2.7mM KCl, 8mM Na2HPO4 and 1.5mM KH2PO4, pH7.4) and fixed with 3.7% paraformaldehyde at room temperature for 20 minutes. The fixed cells were washed with PBS for three times and then treated with 0.1% Triton X-100 for 2 minutes. After three times washing with PBS, mouse anti-FAKp (1:100 in PBS) and rabbit anti-plectin (1:100 in PBS) antibody were simultaneously added to the pre-treated cells for 1 hour. For fluorescent imaging, cells were incubated with secondary antibodies (Cy2-conjugated anti-mouse IgG and Cy3-conjugated anti-goat IgG) and Cy5- conjugated Phalloidin dye (1:100 in PBS) for 1 hour. After three-time washing with PBS, fluorescent images were viewed under the Zeiss LSM 510 confocal microscope.
The paraffin-removed sample sections were rehydrated and treated with 3% H2O2 for 10 minutes to eliminate the endogenous peroxidase activity. Non-specific binding were blocked with bovine serum albumin for 10 minutes. The sample sections were incubated with anti-plectin (1:40 dilution) and anti-FAKp (1:50 dilution) monoclonal antibodies for 1 hour at room temperature. The biotinylated anti-goat and anti-mouse IgGs were then added (1:400 dilution) and incubated at room temperature for 1 hour. The specific bindings were detected by adding avidin-conjugated peroxidase and observed under a light microscope (BX51; Olympus, Tokyo, Japan) in presence of the substrate reagents. For each HCC specimen, the immunohistochemistry assay was triplicated on separate sample sections.