Evaluation of the putative lymphoma-associated point mutation D427H in the STAT3 transcription factor

Plasmids, mutagenesis and cell culture

This study was performed using two expression plasmids, namely pSTAT3-GFP encoding a carboxy-terminal fusion protein of the full-length murine STAT3 cDNA with green fluorescent protein (GFP) and pSTAT3-SNAP with a SNAP tag [10, 18]. The two vectors were received as a kind gift from Prof. Gerhard Müller-Newen from the Universitätsklinikum RWTH Aachen, Germany. The QuikChange II kit from Stratagene was used to perform site-directed mutagenesis to introduce point mutations in the expression plasmids. For this purpose, we used the following primers with mutated codons underlined (only forward primers are shown):

D427HF; 5´- GGAGGCCGTGCCAATTGTCATGCCTCCTTGATCGTGACTG -´3,

F174AF; 5´- CTCCAGGACGACTTTGATGCCAACTACAAAACCCTCAAG -´3,

All point mutations were confirmed by standard dideoxy-termination DNA sequencing (Microsynth Seqlab, Göttingen) and the generated vectors were used for transfection experiments. In addition to HeLa-S3 cells, STAT1-negative U3A cells were transfected with the corresponding plasmids in order to control for the interference of co-expressed STAT1 on STAT3 signal transduction [19]. These cell lines were a kind gift from Prof. Uwe Vinkemeier, University of Nottingham, United Kingdom. The cells were cultured in a humidified 5% CO2 atmosphere at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) (for U3A cells; PAA Laboratories) or Roswell Park Memorial Institute 1640 medium (RPMI) (for HeLa cells; Lonza) supplemented with 10% fetal calf serum (FCS; Biochrom), 100 IU/ml penicillin, 100 IU/ml streptomycin, and 0.04 μg/ml puromycin (only for U3A cells; Sigma-Aldrich). Cells were transfected with MegaTran2.0 (Origene) and on the next day stimulated with 25 ng/ml of recombinant human IL-6 (Gibco) or 50 ng/ml of recombinant human IFNγ (Biomol) for the indicated times. For EMSA extracts, STAT3-variant expressing cells were stimulated for 30 min with the cytokines indicated.

Protein extraction and detection

Cells expressing recombinant STAT3 tagged with either GFP- or SNAP were grown on 6-well dishes and lysed on ice for 5 min in 50 µl cytoplasmic extraction buffer (20 mM HEPES, pH 7.4, 10 mM KCl, 10% (v/v) glycerol, 1 mM EDTA, 0.1 mM Na3VO4, 3 mM 1,4-dithiothreitol [DTT], 0.1% IGEPAL-CA-360, 0.4 mM Pefabloc [Sigma-Aldrich], and Complete Mini protease inhibitors [Roche]). The lysates were centrifuged for 15 s at a temperature of 4 °C and 16,000 g. The supernatants were spun again for 5 min and collected as cytoplasmic extracts. The pellets reserved from the first centrifugation step were resuspended in a volume of 50 µl nuclear extraction buffer (20 mM Hepes, pH 7.4, 420 mM KCl, 20% (v/v) glycerol, 1 mM EDTA, 3 mM DTT, 0.1 mM Na3VO4, 0.4 mM Pefabloc, and Complete Mini protease inhibitors) and incubated for 30 min on ice. Subsequently, these samples were centrifuged for 15 min and 4 °C at 16,000 g and collected as nuclear extracts. Equal amounts of nuclear and the corresponding cytoplasmic extracts were mixed for each sample. The combined extracts were then boiled for 3 min in sodium dodecyl sulphate (SDS) sample buffer and resolved by 10% SDS–polyacrylamide gel electrophoresis (PAGE) with subsequent transfer onto poly-vinylidene difluoride (PVDF) membranes. The membranes were then blocked with 25% bovine serum albumin in Tris-buffered saline with 0.05% Tween-20. These blots were then incubated with either the monoclonal phospho-Tyr705-specific STAT3 antibody D3A7 or the monoclonal pan-STAT3 antibody D1B2J, both obtained from Cell Signaling, and after three washing steps exposed to the conjugated secondary anti-rabbit IRDye 800CW antibody (LI-COR). Bound immunoreactivity on the membrane was detected using the LI-COR Odyssey imaging system. The pSTAT3 band intensity was normalized to the corresponding amount of total STAT3 for each variant and stimulation condition.

Fluorescence microscopy

Direct fluorescence microscopy was employed to monitor the kinetics of IL-6- and IFNγ-induced nuclear accumulation of STAT3 variants in U3A and HeLa cells [18]. Cells expressing GFP- or SNAP-tagged STAT3 grown in 8-well chamber slides were stimulated with cytokines, as indicated. SNAP-tagged STAT3 was visualized by staining the cells for 30 min with the SNAP-tag substrate SNAP-Cell TMR-Star from New England Biolabs. Thereafter, cells were washed thrice with pre-warmed culture media and rested for 30 min in their media before being stimulated with cytokines. At the end of stimulation, cells were fixed for 15 min with 4% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature and subsequently nuclei were stained with 5 μg/ml of Hoechst dye 33258 (Sigma-Aldrich) for 10 min. For antibody staining, cells expressing GFP- or SNAP-tagged STAT3 were, after cytokine stimulation, fixed with methanol for 15 min at -20 °C and subsequently permeabilized with 1% Triton X-100/PBS at room temperature (RT) for 20 min. To saturate unspecific binding sites, the cells were treated with 25% FCS in PBS for 45 min while being shaken. This was followed by a 45-min shaking at RT with the primary antibody (monoclonal rabbit phospho-Tyr705-specific STAT3 antibody, Cell Signaling, D3A7; 1:1000 in 25% FCS/PBS). The cells were then washed thrice with PBS. For the detection of the primary phospho-STAT3 antibody, a Cy3-coupled anti-rabbit IgG secondary antibody from goat (Jackson Immunoresearch Laboratories, USA; 1:1000 in 25% FCS/PBS) was added and incubated for 45 min at RT while being shaken, followed by nuclear staining. Slides were mounted in a fluorescence mounting medium, which was obtained from Southern Biotech. Intracellular fluorescence staining patterns were visualized using a Nikon Eclipse Ti fluorescence microscope, which was equipped with appropriate filters. Images taken with a Nikon DS-Qi2 camera were further processed with the NIS elements software (Nikon). Nuclear and total cellular fluorescence intensities were determined using ImageJ (NIH) and normalized to the background intensity of the respective images. Mean nuclear-to-total cellular fluorescence intensity ratios including their standard deviations were calculated from 20 randomly selected transfected cells, for each variant.

Electrophoretic mobility shift assay

STAT3 proteins were examined by means of electrophoretic mobility shift assays (EMSA) for their binding to specific or mutant duplex oligonucleotides containing a single sis-inducible element (SIE)/GAS element. Cellular extracts (4 µl) from IL-6- or IFNγ-stimulated cells expressing recombinant STAT3 were incubated with EMSA reaction buffer (8 µl) containing the unspecific, synthetic competitor poly(deoxyinosinic-deoxycytidylic) acid and 1 ng of the [33P]-labelled duplex oligonucleotide probe, which was generated by an end-filling reaction using the Klenow fragment (New England Biolabs). A duplex oligonucleotide M67 with a single, canonical SIE/GAS site was used to test the binding of STAT3 variants (SIE/GAS site is underlined, anti-sense oligo is not shown): 5´-TTTTCGACATTTCCCGTAAATCTG-´3. To test for changes in sequence-specific DNA binding of the STAT3 mutants, additional oligonucleotides with two complete GAS sites in tandem orientation (2xGAS) or one complete GAS site (GAS-nonGAS) or no GAS site (2xnon-GAS) were used:

2xGAS; 5′-TTTTCGTTTCCCCGAAATTGACGGATTTCCCCGAAAC-′3,

GAS-nonGAS; 5′-TTTTCGTTTCCCCGAAATTGACGGATTTACCCCAAC-′3,

2xnonGAS; 5′-TTTTCGTTTACCCCAAATTGACGGATTTACCCCAAC-′3.

For competition experiments, cellular extracts were incubated with [33P]-labelled duplex M67 oligonucleotides for 15 min at RT in EMSA buffer. Subsequently, a 750-fold molar excess of unlabelled M67 DNA was added for 10 min on ice. The reactions were loaded on a 4.8% acrylamide:bisacrylamide (29:1) gel at 4 °C and separated at 400 V. Sequence-specific DNA binding was visualized on vacuum-dried gels using the laser phosphorimaging system Typhoon FLA 9500 (GE Healthcare Life Sciences). The band intensity corresponding to the STAT3 variants on the autoradiograms was either measured as an absolute value or divided by the intensity of a fast-migrating unspecific band, which was regarded as a loading control and is labelled with an asterisk in the figures.

Reporter gene assays and real-time PCR

Promoter activation mediated by STAT3 was studied in transfected U3A cells using a vector encoding a luciferase reporter with three copies of an IFNγ-inducible Ly6E GAS element in the promoter region upstream of the transcriptional start site [20, 21], as described in our previous paper [18]. U3A cells grown on 48-well plates were co-transfected with three vectors in each well: the luciferase reporter 3xLy6E (70 ng), a β-galactosidase plasmid (200 ng), and an expression plasmid encoding for either GFP- or SNAP-tagged WT or mutant STAT3 (250 ng). Twenty-four hours after transfection, cells were either untreated or treated with IL-6 or IFNγ for 6 h, before cellular extracts were prepared using a lysis buffer containing 25 mM glycylglycine, 1% Triton X-100, 15 mM MgSO4, 4 mM EGTA, 0.4 mM Pefabloc, 3 mM DTT, pH 7.8, and Complete protease inhibitors. Reporter gene expression was assessed by means of a luciferase assay system from Promega using the luminometer Centro KS LB960 (Berthold Technologies). Luciferase expression was first normalized to the β-galactosidase activity, which was determined spectroscopically at a wavelength of 420 nm in the corresponding samples. Five independent transfections were tested for each STAT3 variant and stimulation mode, and the experiment was repeated in duplicate.

To test endogenous target gene induction, U3A cells expressing STAT3-GFP were cultured for 18 h in Dulbecco’s modified Eagle’s medium supplemented with 1% FCS, prior to stimulation for 3 h with IL-6 or IFNγ. RNA was isolated from the cells using the peqGold Total RNA kit (VWR Lifesciences). First-strand cDNA synthesis was carried out using the Verso cDNA Synthesis kit (Thermo Fisher Scientific). Real-time PCR reactions were performed in a volume of 20 µl, containing 25 ng cDNA, 70 nM of each specific primer, and 10 µl of Absolute Blue qPCR SYBR Green Mix (Thermo Fisher Scientific). The following primer pairs were used, according to sequence data available from the National Center for Biotechnology Information (NCBI):

hCyclinD1F; 5'-CGG TGT CCT ACT TCA AAT GT-3',

hCyclinD1R; 5'-ATG GAG TTG TCG GTG TAG AT-3',

hc-MycF; 5´-GGTCTTCCCCTACCCTCTCAACGA-´3,

hc-MycR; 5´-GGCAGCAGGATAGTCCTTCCGAGT-´3,

hGAPDHF; 5´-GAAGGTGAAGGTCGGAGTC-´3, and.

hGAPDHR; 5´-GAAGATGGTGATGGGATTTC-´3.

The PCR protocol included a denaturation step at 95 °C for 15 min and 40 cycles of denaturation at 95 °C for 15 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s. After the final amplification step, a melting curve analysis was performed on the Eppendorf Mastercycler ep realplex 2 using a temperature gradient from 60 °C to 95 °C in 0.5 °C increment steps and fluorescence being measured at each temperature for a period of 10 s. The relative expression of a target transcript was normalized to the expression of the GAPDH gene. The △△ Ct-method was used to determine comparative relative expression levels, based on the formula 2−(△Ct target − △Ct reference sample). All real-time PCR reactions were performed in two independent transfection experiments with duplicate reactions each.

Data analysis

ImageJ (NIH) software was used to process digital images and data figures were created using CorelDRAW Graphics Suite 2019. Means and standard deviations were calculated for each STAT3 variant and stimulation condition. Data were analysed using the program GraphPad PRISM. Differences between the groups were assessed using Student’s t-test, and a p-value ≤ 0.05 was considered to indicate statistical significance.

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