Cell culture and stimulation with IL-1 and IL-6
HepG2 cells (ATCC) were cultured at 37°C and 5% CO2 in Dulbecco Modified Eagle Medium (DMEM) with 1000 mg/L D-glucose (Sigma-Aldrich), supplemented with 10% foetal bovine serum (Gibco/BRL). Cells were grown to approximately 80% confluence. Fifteen hours before cytokine treatment they were washed twice in PBS and placed in DMEM containing 0.5% FBS. Then the cells were stimulated with human recombinant IL-1 (BioMol) at the final concentration of 15 ng/ml, or with human recombinant IL-6 (MP) at the final concentration of 25 ng/ml, or with a mixture of both. In the case of experiment with IL-1 or IL-6 applied separately, cells were stimulated for 4, 12, and 24 h. When both cytokines were used, cells were prestimulated for 3 h with IL-1 and then stimulated with IL-6 for the time indicated. The concentration of both cytokines was optimized experimentally. The responsiveness of HepG2 cells to these cytokines was evaluated in each series of experiments by comparing changes in the levels of mRNA coding for known acute phase proteins . Non-stimulated cells harvested after 4, 12, and 24 h of the experiment served as controls.
Generation of genetic constructs and transfection procedure
The insert for pcDNA3mim and pQE31mim vector, containing complete coding region of mimitin gene, was generated by PCR in two steps. The first round of PCR was carried out with forward primer: tgctggcagcgctggaaac and reverse: agtcacatccatatacatgaaaag. The following nested primers: forward: cggcatgggttggtctc with restriction site for KpnI and reverse: atgcattcattgattgtcgc with restriction site for SmaI/XmaI were used to obtain a region of 520 nucleotides, corresponding to positions -4 to 516 relative to the mimitin ATG start codon. After digestion of the PCR product with KpnI/SmaI and of the pcDNA3 vector with KpnI/EcoRV ligation was carried out. For the recombinant protein purification the same procedure was used but the forward primer had SacI restriction site. Both constructs were sequenced before the transfection/transformation experiment. Plasmids overerexpressing MAP1S were kindly provided by Prof. Friedrich Propst . Expression of MAP1S was under the control of tet-responsive promoter. For overexpression of MAP1S in HepG2 cells co-tranfection of UHD15-1 (containing tTA transactivator) and pUHD10-3 (containing full length MAP1S) was carried out. To overexpress mimitin or MAP1S, HepG2 cells were transfected with the following genetic constructs: pcDNA3 empty vector (control), pcDNA3mim, pUHD15-1 or pUHD10-3 using Lipofectamine 2000 (Invitrogen) according to the manufacturer's procedure.
To estimate the efficiency of transfection in different samples we performed RT-PCR using primers specific for a transcript coding for aminoglycoside phosphotransferase (neomycin resistance gene; coded by pcDNA3) and EF2 as a reference gene (NeoF: tgctcctgccgagaaagtat and NeoR: aatatcacgggtagccaacg; EF2 11: gacatcaccaagggtgtgtgcag and EF2 22: gcggtcagcacaatggaata).
Northern blot analysis
In order to analyse tissue distribution of the mimitin transcript a commercial blot filter was used (Clontech). The membrane had been loaded with 2 μg of mRNA isolated from the following human tissues: brain, heart, skeletal muscles, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung and peripheral blood leukocytes. Prehybridization and hybridization were carried out at 65°C in 1% SDS, 1 M NaCl, 10% dextran sulfate solution. Thirty nanograms of the probe was labelled with [α-32P]dCTP using a random primer labelling kit (Promega). After washing (20 min at room temperature in 2 × SSC, 20 min at 65°C in 2 × SSC and then twice for 20 min. at 65°C in 1 × SSC/1% SDS) the RNA blot was read using a Molecular Imager FX and analysed with Quantity One (Biorad). The blot was later stripped and reprobed with a β-actin probe provided with the kit to check for equal loading.
In the experiment where the changes in mimitin transcript level were studied total RNA was isolated from control (unstimulated) cells and from cells after IL-1 and IL-6 treatment. The guanidinum/phenol extraction procedure was used. Ten micrograms of total RNA was separated in 1% agarose-formaldehyde gel and blotted onto a nitrocellulose membrane. Prehybridization and hybridization were carried out as above. Quantitative analysis of the transcript was based on densitometric readings from Quantity One software. The intensity of each analysed band was divided by the intensity of the 28S rRNA band and the final results were calculated as the multiple of suitable control. The reported values represent the mean of three independent experiments.
Generation of mimitin-specific monoclonal and polyclonal antibodies
E. coli BL21 cells were transformed with pQE-31 vector containing the entire coding sequence of mimitin. Recombinant protein was purified from bacterial culture using BD Talon Metal Affinity Resins with cobalt ions (BD Biosciences) and used in immunization of New Zealand White rabbit (3 times 300 μg of antigen). Antibodies were purified from rabbit sera by protein A-Sepharose chromatography and then checked for specificity. The antibodies were analyzed with protein extracts from HepG2 cells and with recombinant mimitin by Western blotting. There was no cross-reactivity and only specific product of the expected size was observed on the membrane. An additional proof of the quality and specificity of the obtained antibodies is the observed reduction of the signal in the Western blot after the cells were transfected with siRNA specific for mimitin.
For generation of a monoclonal antibody Balb/c mice were immunized several times with recombinant mimitin according to a standard protocol . The specific IgG titre exceeded 1:105. Splenocytes were fused with Sp2/0 myeloma cells (HPRT-/-) using polyethylene glycol (Sigma-Aldrich) and the mixture of cells was cultured for two weeks in HAT selection medium enriched with 10% FCS and hybridoma growth supplement (Sigma-Aldrich). The clones were analyzed for the synthesis of anti-mimitin Ab in direct ELISA on microplates covered with recombinant mimitin (2 μg/ml). The clone 1F5D8 obtained after a two-step subcloning procedure performed by serial dilution was chosen for further work. Undiluted culture medium of this clone (DMEM + 10%FCS) was used as a source of the detecting antibody in indirect ELISA.
Cell lysate preparation and Western blot analysis
Whole cell protein extracts were obtained by washing cells twice with ice-cold PBS and harvested in 1 ml of PBS containing 10% (v/v) glycerol and EDTA (5 mM). After centrifugation (1000 g, 10 min, 4°C), cell pellets were suspended in 50 μl of lysis buffer (100 mM Tris-HCl pH 7.5; 1% Triton-X100 and protease inhibitor cocktail (Roche)). Protein concentration of each sample was determined using the bicinchoninic acid assay (BCA, Sigma-Aldrich). Ten micrograms of protein extract was loaded onto 10% polyacrylamide gel and separated by SDS/PAGE in Tris-tricine buffer . After transfer to nitrocellulose membrane blots were blocked with 2% BSA in TBS-N buffer (20 mM Tris pH 8.0, 150 mM NaCl, 0.05% Nonidet P-40) overnight at 4°C and then incubated with primary antibodies for 2 h at room temperature. Polyclonal antibodies against mimitin, generated as described above, were used at 3 μg/ml. Secondary HRP-conjugated anti-rabbit IgG was used at 1:80 000 dilution (Sigma-Aldrich). Both primary and secondary antibodies were diluted with TBS-N containing 2% BSA. TBS-N was used for washing the membranes before, between and after incubation with antibodies. Immunoreactive bands were detected by an enhanced chemiluminescence system (SuperSignal West Pico Chemiluminescent Substrate, Pierce) and visualized on medical x-ray film.
Identification of signalling pathway
HepG2 cells stably transfected with retroviral vector pCFG5-IEG2 encoding a non-degradable mutant form of IκBα, and cells with an empty vector (control) were used to determine of the NFκB signalling pathway in IL-1-dependent activation of mimitin gene. These cells were kindly provided by Professor Stephan Ludwig (Heinrich-Heine University, Duesseldorf, Germany; ).
For determination of MAPK signalling pathway involvement in mimitin gene activation HepG2 cells were serum-starved overnight in 0.5% FBS in DMEM. Then the cells were pre-treated with MAPK inhibitors: 10 μM U0126 (Calbiochem), 10 μM SP600125 (Calbiochem), or 10 μM ZM336372 (Calbiochem) for 30 min and stimulated with15 ng/ml IL-1 for 12 h. Mimitin transcript level was measured by real time PCR. The inhibitors' activity was controlled by analysis of phosphorylation of MAP kinases (Western blot). For control experiment cells were also pre-treated with the inhibitors and stimulated with 15 ng/ml IL-1 for 10 min. Cell lysates were analyzed by Western blotting with anti-P-Erk and anti-P-JNK (Cell Signaling Technology) and anti-P-p38 antibodies (Abcam).
Real Time PCR (Q-RT-PCR)
cDNA was synthesized from 1 μg of total RNA in 20 μl using SuperScript RNaseH- reverse transcriptase (Promega) and oligo (dT) primer (Promega). Real time PCR was carried out using the SYBR Green PCR Master Mix (DyNAmo™ HS SYBR Green qPCR (Finnzyme), 1 μl of 5× diluted cDNA, and 20 ng of each primer. For mimitin transcript the forward primer was 5'-ctgcctccaccagttcaaactc-3' and the reverse primer was 5'-gtcgcatccatatacgtgaaaag-3'. The amount of each transcript, expressed as fold-variation over control (untreated cells), was calculated after determination of the difference between the CT of the transcript studied and the calibrator transcript for elongation factor 2 (EF2). The CT values used were means of triplicate measurements.
Subcellular localization of mimitin
For subcellular fractionation of HepG2 cells the Qproteome Kit (Qiagen) was used according to manufacturer's procedure. Consecutive centrifugation and extraction with suitable buffers allowed the isolation of cytosolic, membrane, nuclear and cytoskeletal proteins. Protein concentration was determined with BCA protein assay. Additionally, the mitochondrial fraction was isolated as described by Ndubuisi and co-workers . Ten micrograms of protein extracts from each fraction was used in Western blot analysis. For detection of individual proteins different polyclonal antibodies were used (Abcam): for the nuclear fraction – against laminin, for the cytosolic fraction – against GAPDH, for the membrane fraction – those against prohibitin.
Mimitin levels in control and cytokine-stimulated HepG2 cells were measured using sandwich ELISA. A 96-well microtiter plate was coated with rabbit polyclonal antibodies against mimitin. To each well 100 μl of antibodies (6 μg/ml) diluted in 50 mM borate buffer (pH 8.35) was added. Plates were incubated overnight at 4°C. After washing five times with PBS a protein standard (6, 12, 25, 50, 100, 200, 400, 800 pg/ml in 0.5% BSA in PBS) and cell lysates (2.5 μg) were added in duplicates. Incubation for 1 h at 37°C was followed by washing with PBS to remove unbound proteins. Subsequently, plates were incubated with biotin-labelled anti-mimitin antibodies (1 μg/ml in 0.5% BSA in PBS) for 1 h at 37°C. Then, to each well 50 μl of avidin-HRP (Sigma) (1:25000 in 0.5% BSA in PBS) was added and incubation was carried out for 30 min at 37°C. Each incubation step was followed by extensive washing with PBS. The enzymatic reaction was performed using the TMB Substrate Reagent Set (BD Biosciences), terminated by adding 50 μl 1 M HCl and the absorbance was measured at 450 nm. The values represent the mean ± SD of three independent experiments performed in duplicates.
Pre-designed siRNA obtained from Ambion included GAPDH siRNA as a positive control, and siRNA with scrambled sequence as a negative control. The two different siRNA sequences specific for mimitin were as follows: (A) gaaaucaaaauaaaaagccuu and (B) gccacaaucaaugaaugcatt.
HepG2 cells were cultured in 12-well plates as described above. The siRNA concentration and the amount of transfection reagent were optimized experimentally. Before transfection, cells were trypsinized, centrifuged at 1000 rpm at 4°C for 5 min and resuspended in fresh medium. Lipid-based transfection reagent siPORT NeoFX (4 μl/well) from Ambion and siRNAs (50 nM final concentration) were separately diluted in 50 μl OPTIMEM and then mixed together. Transfection complexes were allowed to form for 10 min and overlayed with 9 × 104 cells per well. As an additional control, some wells were treated with transfection reagent only. The silencing of mimitin was confirmed by Western blotting in three independent experiments.
Caspase activity assay
To analyse caspase 3/7 activity, HepG2 cells were transfected with mimitin-specific siRNA, GAPDH-specific siRNA or negative control siRNA as described before. After 72 h incubation, apoptosis was induced in some of the wells with 15 ng/ml TNFα (PromoCell) and cycloheximide at the final concentration of 40 μM per well. Cells were scraped in RIPA buffer (25 mM Tris HCl pH7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) when most of the stimulated cells in a well showed signs of apoptosis before complete detachment (after 5 h of treatment). Apoptosis was assessed by analysis of activation of caspase-3 and -7 using the DVD-aminoluciferin substrate from the Caspase-GloTM3/7 assay kit (Promega). After protein isolation, 5 μg of protein from each well was diluted in RIPA buffer to a final volume of 50 μl and added to an equal amount of Glo-reagent. Reaction mixture was incubated for 1 h at room temperature in the dark. Caspase 3/7 activity was measured using luminometer (MicroLumat LB96P; EG&G Berthold).
HepG2 cells were transfected with siRNA specific for mimitin, GAPDH or negative control siRNA in a 96-well plate. In each well 800 cells were seeded and the ratio of transfection mixture to cell density was the same as in previous experiments. Additionally, cells were transfected in a 12-well plate for Western blot analysis. BrdU-labelling solution from the Cell Proliferation ELISA kit (Roche Applied Sciences) was diluted 1:2000 and added to the 96-well plate 54 h post transfection. After 24 h of labelling, protein fraction was isolated from 12-well plates and medium was removed from 96-well plates. Incorporated BrdU was detected via ELISA with anti-BrdU-POD antibody diluted 1:200 according to the manufacturer's protocol.
Yeast two-hybrid analysis
For yeast two-hybrid screening the Matchmaker human foetal brain cDNA library was used according to manufacturer's procedure (Clontech). The 510-bp fragment encoding full length cDNA of mimitin was generated by RT-PCR using primers:
F – atgggttggtctcagga with restriction site for NdeI, and R-atgcattcattgattgtggct with restriction site for BamHI. After digestion with NdeI/BamHI the PCR fragment was cloned into pGBKT7 vector of the same restriction sites. The correctness of the cDNA sequence was confirmed by sequencing. Before the cDNA library screening, two control experiments were carried out: to verify whether the pGBKT7-mimitin vector expresses the protein in the AH109 strain of Saccharomyces cerevisiae, and to find out whether the "bait" (mimitin) does not autonomously activate the reporter genes. Expression of the fusion protein Gal4-mimitin was verified by Western blotting with monoclonal antibodies specific for c-Myc. Two-hybrid screening was carried out by mating the AH109 yeast transformed with pGBKT7-mimitin (bait) and Y187 yeast transfected with human foetal brain cDNA library in pACT2 vector. Positive and negative controls for the experiment were carried out in accordance with the protocol. A portion of the mating culture in the appropriate dilution was plated on SD/-trp, SD/-leu and SD/-trp,-leu selection medium to calculate the number of screened cells and determine the mating efficiency. Altogether, we screened 6 × 105 clones and achieved 8% mating efficiency. The rest of the mating culture was spread on a plate with SD/-trp,-leu,-his,-ade medium to select colonies containing both bait and prey. Plasmids isolated from positive clones were purified from yeast and those containing "prey" were amplified in E. coli and co-transformed to yeast with the original bait (pGBKT7-mimitin) to verify specific activation of the reporter genes (HIS3 and lacZ) and the sequences compared with NCBI data.
HepG2 cells were transfected with the pcDNA3mim plasmid encoding mimitin or with pUHD10-3 encoding Myc-tagged MAP1S and its transactivator pUHD15-1, or co-transfected with all these vectors using Lipofectamine 2000 (Invitrogen). The coimmunoprecipitation analysis was performed 24 h after transfection with the ProFoundTM c-Myc Tag IP/Co-IP Kit (Pierce) following the manufacturer's protocol. Cell lysates and obtained eluates were loaded on 10% SDS-PAGE for Western blotting. Proteins were detected with antibodies specific for mimitin and for the Myc tag.
The data from real-time PCR, Northern blotting, measurements of caspase activity and of proliferation rate were examined for statistical significance using Student's t test. A p value less than 0.05 was taken as statistically significant.