Glioblastoma is associated with poor survival and a high recurrence rate in patients due to inevitable uncontrolled infiltrative tumor growth. The elucidation of the molecular mechanisms may offer opportunities to prevent relapses. In this study we investigated the role of the activating transcription factor 3 (ATF3) in migration of GBM cells in vitro. RNA microarray revealed that gene expression of ATF3 is induced by a variety of chemotherapeutics and experimental agents such as the nitric oxide donor JS-K (O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate). We found NFκB and STAT3 to be downstream targets inhibited by overexpression of ATF3. We demonstrate that ATF3 is directly involved in the regulation of matrix metalloproteinase expression and activation. Overexpression of ATF3 therefore leads to a significantly reduced migration capacity and induction of tissue inhibitors of matrix metalloproteinases. Our study for the first time identifies ATF3 as a potential novel therapeutic target in glioblastoma.
Glioblastoma multiforme (GBM) is the most frequent and malignant brain tumor with few therapeutic options and a poor clinical outcome. The understanding of the mechanisms that drive tumor progression plays a central role in the current GBM research, while elevated invasive capacity and multifocal growth are of great importance from a clinical point of view. Understanding the molecular regulatory mechanisms is therefore fundamental for the development of novel anti-tumor therapies to achieve tumor elimination. The ability to degrade extracellular proteins is essential for any individual cell to interact properly with its immediate surroundings. For multicellular organisms it is a prerequisite for development and chemotaxis.1 In physiological conditions, the extracellular matrix (ECM) is involved in wound healing and organogenesis; in pathological conditions it is significantly involved in local tumor progression and metastasis in cancer.2,3 Migration and metastasis are complex interactions between cells and the ECM. There are various groups of proteolytic enzymes or proteases involved in matrix degradation, but the matrix metalloproteinase (MMP) group of enzymes is the most important in the context of tumor invasion and metastasis.2,4 The MMPs are zinc and calcium-dependent endopeptidases that modify signaling pathways and cell functions under physiological and pathological conditions.5 MMPs are involved in migration, inflammation, proliferation, apoptosis and differentiation and are therefore latently synthesized.5,6 The substrates of MMPs are specific proteins of the ECM like collagen, fibrin, integrins and receptors.2,7 Various studies demonstrated a correlation between expression of MMPs and the malignancy and invasive capacity of GBM.8 Certain MMPs such as MMP1 and MMP7 are associated with a poor prognosis in cancer.5,9,10 MMPs are secreted in the ECM as inactive zymogens interacting with tissue inhibitors of metalloproteinases (TIMP).4 TIMPs bind to the catalytic domain of MMPs and can inhibit the ‘cysteine-switch’, which disrupts the interaction between a cysteine in the prodomain and the Zn2+ ion in the active site. After the cleavage of the complex the prodomain of the MMP is released for activation.11,
The main objective of this study was to investigate the molecular mechanisms of migration induced by low dose NO released from the diazeniumdiolate JS-K in glioma cells in vitro. ATF3 was found to be upregulated by NO in RNA microarray. In this study we provide evidence that overexpression of ATF3 correlates with expression and activation of MMPs and TIMPs in vitro. The reduced activation of MMPs leads to restricted migration capacity of established and primary GBM cells in vitro. In addition, we demonstrate a positive correlation between ATF3 and phosphorylation of STAT3 and the inhibition of nuclear translocation of NFκB in glioblastoma cells. We show that the transcription factor ATF3 directly regulates gene expression of nfκb, stat3 and klf6 that are involved in tumor progression and invasion. Therefore, we propose that ATF3 could serve as prognostic marker for migration and metastatic disease in glioblastoma.
Nitric oxide reduces migration capacity in a time- and dose-dependent fashion
To investigate the influence of JS-K on migration capacity of glioma cells, we performed a wound closure assay over 96 h for U87 and primary IC cells (Figure 1). Starting at 24 h, the ability to close the migration gap is time-dependently reduced in both cells lines. In U87 cells, this significantly reduced effect can be observed at a concentration of 2 μM JS-K (Figure 1a), whereas in primary IC cells it emerged at 1 μM (Figure 1b). Up to 3 μM JS-K progressively inhibited migration in a dose-dependent manner.
Migration capacity is not influenced by reduction of viability and proliferation
One explanation for cells not closing a wound can be increased cell death elicited by the treatment or the inhibition of proliferation. To exclude these factors caused by JS-K we investigated cell viability by MTT assay over 96 h as well as the proliferation rate by BrdU incorporation assay (Figures 1c–f). Less than 20% of U87 cells were killed at the highest dose of 3 μM after 96 h (Figure 1c). Viability of primary IC cells was not affected by JS-K up to 72 h after treatment (Figure 1d). At 96 h, the viability decreased to 68% when using 3 μM JS-K. In contrast, no influence of JS-K on proliferation was detected at the tested doses at all (Figures 1e and f). At no time point either cell line showed a significant reduction or inhibition of proliferation.
Microarray analysis reveals activating transcription factor 3 to be upregulated by NO
To identify the gene targets of NO, we determined the temporal gene expression profile of U87 cells exposed to NO for 48 h using RNA microarray. We used U87 cells as a test cell line because this cell line is well characterized for many years as a standard for biochemical and genetic experiments in glioblastoma research. The data set was analyzed for upregulated and downregulated genes more than 2-fold in a statistically significant manner (P≤0.05). Approximately 10 genes were found to be upregulated (Table 1) and further analyzed by qRT-PCR to validate the array (data not shown). Activation transcription factor 3 (ATF3) was found to be upregulated 2.51-fold in the microarray. Primary IC cells and U87 cells revealed dose-dependent upregulation of expression in qRT-PCR after 48 h exposure to JS-K at concentrations up to 15 μM (Figure 2). U87 cells showed a 15-fold expression compared to controls at the highest concentration of 15 μM (Figure 2a). Primary IC cells had the highest peak of 5.8-fold expression when using 10 μM JS-K (Figure 2b). ATF3 is known for its key role in suppression of metastasis and invasion. Therefore we chose migration for further investigation of the role of ATF3 in glioblastoma. To demonstrate the relevance of ATF3 we examined ATF3 RNA expression in response to treatment with various anticancer agents (Figure 2c). Epidermal growth factor (EGF) and hydrogen peroxide (H2O2) which is known to induce necrosis, did not induce the expression of ATF3 more than 2-fold. Temozolomide (TMZ), a common chemotherapeutic agent used in GBM therapy, did not even induce a twofold upregulation in contrast to Etoposide (ETO), also used for GBM therapy, leading to a 16-fold expression of ATF3. Paclitaxel (Taxol), a chemotherapeutic for a variety of tumors, showed a 9.5-fold expression and the anti-inflammatory acetylsalicylic acid (ASA) a 3.4-fold expression of ATF3. This highlights the considerable importance of research into ATF3 in glioblastoma.
Overexpression of ATF3 reduces migration capacity and proliferation in vitro
Since we observed the overexpression of ATF3 in GBM cells induced by NO as well as different anticancer agents, we overexpressed and knocked down ATF3 stably by lentiviral infection in U87 cells. The efficacy of the knockdown (shATF3) and the overexpression (ATF3), done with their controls, pGIPZ and pLOC, are shown in Figures 3a and b. Knockdown, overexpression and treatment with JS-K are normalized to the particular control set to one. The controls still react to JS-K as the uninfected cells (U87). The knockdown of ATF3 led to a decreased ATF3-expression to 25% compared to control. The overexpression showed RNA levels of more than 650-fold compared to control and could be still increased by JS-K. To demonstrate the effect of ATF3 on migration of U87 cells we repeated the wound closure assay. Cells infected with pLOC, pGIPZ and shATF3 did not show any difference in migration capacity compared to uninfected U87 cells over the 120 h observation period (Figures 4a and b). However, ATF3-overexpressing cells migrated significantly slower into the gap (Figures 4a and b). While all cells not overexpressing ATF3 closed the gap within 120 h, the ATF3 overexpressing cells still exhibited a gap of about 1 mm, which accounts for 50% of the initial space (Figure 4b). To reveal an association between reduced migration and reduced viability of ATF3 overexpressing cells, we performed Matrigel invasion assay. Compared to controls, knockdown of ATF3 did not lead to altered invasive capability, whereas the overexpression of ATF3 decreased invasion of the cells significantly (P=0.00004) to 32% (Figures 4c and d). Since ATF3 is upregulated by NO, we expected a different behavior to JS-K in cell death induction, especially of shATF3. Cell lines showed no difference in viability when exposed to JS-K up to 25 μM for 48 h in the MTT assay (Figure 4e). The relative cell proliferation measured by the BrdU incorporation assay indicated a significant reduction in ATF3 cells after 72 h (Figure 4f). Both controls and shATF3 showed the same proliferation rate compared to uninfected U87.
Overexpression of ATF3 does not regulate mRNA expression of migration-related genes but prevents translocation of NFκB and STAT3 phosphorylation
The transcription factor ATF3 is known to regulate the expression of a number of genes. To investigate the relevance of ATF3 for migration we performed qRT-PCR for p53, nfκb1, stat3, p38α and klf6. Figure 5a shows the regulation of these genes in ATF3 overexpressing U87 compared to uninfected cells. Stat3 and klf6 were upregulated more than 2-fold in response to overexpression of ATF3 but not p53, nfκb1 and p38α. However, even if stat3 mRNA expression was upregulated, Western blot revealed that STAT3 is no longer phosphorylated with ATF3 overexpression (Figure 6b). When further treated with up to 5 μM JS-K, we observed a threefold upregulation of nfκb1 mRNA expression after 48 h (Figure 5b) but no difference in p53 (Figure 5c), stat3 (Figure 5d), p38α (Figure 5e) and klf6 (Figure 5f). Since the NFκB pathway plays an important role for migration and invasion of tumor cells, we further investigated the activation of NFκB. Immunocytochemistry in cells treated with 5 μM JS-K for 48 h showed no translocation of NFκB (p65) into the nucleus caused by NO in either cell line, control or overexpressing ATF3 (Figure 6a). p65 could be detected in the cytoplasm with a much higher protein level in NO-treated cells compared to controls. Treatment with TNFα is well known to induce translocation of p65 and was used in this experiment as a positive control of translocation. Control cells translocate p65 after stimulation with TNFα for 6 h but not cells overexpressing ATF3 (Figure 6a). NFκB (p65) remained in the cytoplasm and was barely detectable in the nuclei.
Overexpression of ATF3 regulates protein level of matrix metalloproteinases and tissue inhibitors of metalloproteinases 3
Matrix metalloproteinases (MMPs) stimulate tumor metastasis by the degrading extracellular matrix. In this study we found no visible regulation of RNA expression in PCR caused by NO in MMP2, MMP7 and MMP9, nor in their inhibitors TIMP1 and TIMP3 in either U87 (Figure 7a) or primary IC cell lines (Figure 7b). TIMP2 was slightly reduced by increasing concentrations of JS-K in U87, whereas TIMP4 was dose-dependently enhanced by NO (Figure 7a). In U87 cells with ATF3 knockdown we did not observe any difference in expression of MMP1–3, MMP7 or MMP9 compared to pGIPZ or uninfected controls (Figure 7c). In contrast, cells overexpressing ATF3 exhibited no RNA expression of MMP7 compared to pLOC and uninfected cells. TIMP3 was significantly overexpressed in ATF3 overexpressing cells.
Since MMP2, MMP7 and MMP9 are important for the degradation of the tumor matrix, we investigated their protein levels by Western blot and the activity of MMPs by zymography (Figure 8). Western blot of the conditioned media obtained from U87 and primary IC cells showed a reduced protein level of MMP2, MMP7 and MMP9 with increasing concentrations of JS-K up to 3 μM and an enhanced expression of TIMP3 at 1 and 2 μM in U87 and primary IC cells (Figures 8a and b). The whole cell lysate of U87 cells exhibited an almost equal expression level of MMP2, MMP7 and TIMP3 but a dose-dependently decreasing level of MMP9 (Figure 8c). However, whole cell lysates of primary IC cells revealed decreasing protein levels of MMP2, MMP7 and MMP9 with low dose JS-K and no change in TIMP3 expression (Figure 8d). The protein level of MMP2, MMP7 and MMP9 in the supernatant of ATF3 overexpressing cells was considerably reduced compared to pLOC controls as well as the protein level of TIMP3 (Figure 8e). Western blot of the whole cell lysate of ATF3 knockdown and overexpression indicated a total deficiency of MMP2 and MMP9 in ATF3 overexpressing cells and a lower protein level of MMP7 (Figure 8f). In contrast, the inhibitor TIMP3 was overexpressed in ATF3 cells compared to controls and knockdown (Figure 8f). Zymography analysis of the conditioned media revealed no change in the activation of MMP2 in U87 exposed to NO (Figure 8a), whereas IC cells exhibitied less activation when treated with 3 μM JS-K (Figure 8b). However, ATF3 overexpression reduced the level of active MMP2 in the supernatant of the cells compared to ATF3 knockdown and controls (Figure 8c). MMP7 and MMP9 could not be detected in gelatin or casein gels (data not shown).
Exploration of molecular mechanism in GBMs is of great importance for a full understanding of the biology of this highly malignant cancer and the identification of potential therapeutic targets. Recent research suggest that NO is a putative adjuvant in anti-tumor therapy but some mechanisms remain unclear so far.35,
Materials and Methods
The immortalized human glioma cell line U87MG obtained from ATCC (Manassas, VA, USA) and the primary glioblastoma cell line IC were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum and 1% penicillin/streptavidin at 37 °C in a humidified atmosphere containing 5% CO2. The primary cell line was established from a surgical specimen of a patient (IC) with glioblastoma multiforme. Retrieval and scientific analysis of patient-derived tissue was approved by the local ethics committee under protocol 100020/09. The NO donor JS-K [O2-(2,4-dinitrophenyl)1 [(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate] was synthesized as described earlier.61 Cells were exposed to JS-K concentrations between 0.5 and 25 μM (stock solution 5.2 mM in DMSO) for up to 96 h when they reached 70–80% confluence. The final concentration of DMSO was not higher than 0.05% (when using 25 μM JS-K).
RNA interference and lentiviral transduction
Human shRNAs on lentiviral pGIPZ plasmid targeting ATF3 (V2LHS_172635, V3LHS_405369, V3LHS_352238, V3LHS_405368, V3LHS_352240, V3LHS_405370), overexpression of human ATF3 on lentiviral Precision LentiORF Collection plasmids (pLOC, pLOHS_100010432, pLOHS_100005353)) and negative controls (pGIPZ, pLOC) were transfected into HEK293T cells by cotransfection of lentiviral plasmids pCMV-MD2G and psPAX2 using HiPerFect transfection reagent (Qiagen, Hilden, Germany). Virus containing supernatant was collected and used for infection of U87 cells. shATF3 pLOHS_100010432 was found to knockdown ATF3 expression most efficiently and was used for the experiments as well as V3LHS_405370 for overexpression of ATF3. The functionality of the shRNAs and overexpression plasmids were validated by qRT-PCR analysis. Transduced cells were selected for puromycin resistance before further analysis.
Cells were seeded in 24-well plates with silicon bars to create a gap of about 2 mm. Non-infected cells were treated with JS-K up to 3 μM while cells overexpressing ATF3 or knockdown cells were not treated. After removing the bars cells started migrating from the edge of the wound and repopulated the gap area. The time required for wound closure was measured by microscopy (Zeiss Axiovert, Oberkochen, Germany) and documented up to 120 h.
Boyden chamber assay (BioCoat Matrigel, Corning Inc, NY, USA) was performed to assess the invasion ability of cells overexpressing ATF3 and knockdown. 2×104 cells in serum-free medium were seeded per Boyden chamber and attracted with 10% FBS and 0.02 ng/ml PDGF. Cells were incubated for 21 h at 37 °C in a humidified atmosphere. Invaded cells were fixed in 4% PFA before the surface of matrigels was swabbed to remove non-invading cells. Crystal violet staining (0.2%) was performed according to manufacturer’s protocol. Cells were visualized by microscopy and quantified with ImageJ (National Institutes of Health (NIH), Bethesda, MD, USA; ×10, Zeiss Axiovert). Scale bars represent 500 μm, frames show ×2.5 magnification.
Cell viability was determined by MTT Assay. Cells (104) were grown in 96-well plates with complete DMEM and treated with 1–25 μM JS-K for up to 96 h. MTT Assay was performed as described before.28 Absorbance at 570 nm was measured with Tecan Infinite200 (Tecan, Männedorf, Switzerland). Percentages were calculated relative to the viability of untreated controls set to 100%.
Cell proliferation assay
Cell proliferation was monitored by 5′-bromodeoxyuridine (BrdU) incorporation assay (Roche Diagnostics, Mannheim, Germany). Three thousand cells were seeded in 96-well plates and treated with JS-K up to 3 μM. Cells overexpressing ATF3 or knockdowns were not treated. Cells were stained with BrdU following the manufacturer’s instructions. The percentage of cells exhibiting genomic BrdU incorporation was measured by absorbance at 370 nm with Tecan Infinite200 (Tecan, Männedorf, Switzerland). Percentages were calculated relative to the proliferation of untreated controls or T0.
Gene expression changes were investigated by Microarray using the Illumina HumanHT-12 Expression BeadChip (Illumina Inc., San Diego, CA, USA) for analysis of 31 000 genes. The array was performed at the German Cancer Research Center (DKFZ), Heidelberg, Germany. RNA samples were purified from U87 treated with 15 μM JS-K for 48 h as well as from untreated controls. Gene up- and downregulation caused by JS-K was evaluated. Microarray was done in triplicate.
RNA purification, qRT-PCR and semiquantitative PCR
Total RNA was prepared from U87 and IC cells using the RNeasy mini kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany). cDNA was generated from 1 μg of total RNA in a volume of 30 μl using M-MuLV reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) and 100 pmol of hexameric primers. cDNA was quantified by quantitative real-time PCR on a StepOnePlus System (Thermo Fisher Scientific) using SYBR™ Green Master Mix (Thermo Fisher Scientific) and specific primers for ATF3 (5′- CCTCTGCGCTGGAATCAGTC-3′ forward; 5′- TTCTTTCTCGTCGCCTCTTTTT-3′ reverse), TP53 (5′- AGGGCTCACTCCAGCCACCTG-3′ forward; 5′- AGAATGTCAGTCTGAGTCAGGCCCT-3′ reverse), NFκB1 (5′- AACAGAGAGGATTTCGTTTCCG-3′ forward; 5′- TTTGACCTGAGGGTAAGACTTCT-3′ reverse), STAT3 (5′- CAGGAGGGCAGTTTGAGTCCCTCAC-3′ forward; 5′- GTCGTATCTTTCTGCAGCTTCCGTTCTC-3′ reverse), p38α (5′- CGAGCGTTACCAGAACCTGT-3′ forward; 5′- TGGAGAGCTTCTTCACTGCC-3′ reverse), KLF6 (5′- GGCAACAGACCTGCCTAGAG-3′ forward; 5′- AGGATTCGCTGACATCT-3′ reverse) and RPS18 (5′- TTTTGCGAGTACTCAACACCA-3′ forward; 5′- CCACACCCCTTAATGGCA-3′ reverse) as endogenous control. The conditions were 95 °C for 20 s, followed by 40 cycles of 3 s at 95 °C, 30 s at 60 °C. The relative expression level of the target gene compared with that of the housekeeping gene RPS18 was calculated with the 2−ΔΔCt method and normalized to untreated control set to 1. The semiquantitative PCR was performed with Taq polymerase and buffers provided by Thermo Fisher Scientific. Specific primers were used for MMP1 (5′- GAGCTCAACTTCCGGGTAGA-3′ forward; 5′- CCCAAAAGCGTGTGACAGTA-3′ reverse), MMP2 (5′- GATACCCCTTTGACGGTAAGGA-3′ forward; 5′- CCTTCTCCCAAGGTCCATAGC-3′ reverse), MMP3 (5′- TGAAAGAGACCCAGGGAGTG-3′ forward; 5′- AGGGATTAATGGAGATGCCC-3′), MMP7 (5′- GGCCAAAGAATTTTTGCATC-3′ forward; 5′- GAGCTACAGTGGGAACAGGC-3′ reverse), MMP9 (5′- GCACTGCAGGATGTCATAGG-3′ forward; 5′- ACGACGTCTTCCAGTACCGA-3′ reverse), TIMP1 (5′- AGAGTGTCTGCGGATACTTCC-3′ forward; 5′- CCAACAGTGTAGGTCTTGGTG-3′ reverse), TIMP2 (5′- AAGCGGTCAGTGAGAAGGAAG-3′ forward; 5′- GGGGCCGTGTAGATAAACTCTAT-3′ reverse), TIMP3 (5′- GGTGAAGCCTCGGTACATCT-3′ forward; 5′- AGGACGCCTTCTGCAACTC-3′ reverse), TIMP4 (5′- GGCTCGATGTAGTTGCACAG-3′ forward; 5′- ACGCCTTTTGACTCTTCCCT-3′ reverse) and GAPDH (5′- GGCCTCCAAGGAGTAAGACC-3‘ forward; 5′- AGGGGTCTACATGGCAACTG-3‘ reverse) as endogenous control. A first cycle of 3 min at 95 °C was followed by 30 s at 95 °C, 30 s at 56 °C and 40 s at 72 °C for 40 cycles and finished with 72 °C for 10 min.
U87 and primary IC glioma cells were cultured in DMEM containing 0.4% FBS. Supernatant of JS-K treated cells was collected and frozen at −20 °C. Cells for whole cell lysates were cultured in 10% FBS. Equal amounts of protein (4 μg of supernatant, 20 μg of lysate) were applied on 10% SDS-polyacrylamide gels and electrophoresed (BioRad, Munich, Germany). Proteins were blotted on PVDF-membranes by wet blotting (BioRad, Munich, Germany). Epitopes were blocked with 5% non-fat milk in tris-buffered saline with 0.05% Tween20 for 1 h at room temperature (RT). Blots were incubated with primary antibodies anti-ATF3 (ab87213 1 : 1000 Abcam, Cambridge, UK), anti-MMP2 (#4022 1 : 1000 Cell Signaling Technology, Inc., Danvers, MA, USA), anti-MMP7 (#MAB9071 1 : 1000 R&D System, Inc., Minneapolis, USA), anti-MMP9 (#3852 1 : 1000 Cell Signaling Technology, Inc.) anti-TIMP3 (#D74B10 1 : 1000 Cell Signaling Technology, Inc.), anti-GAPDH (1 : 10000 Abcam) overnight at 4 °C. After incubation with secondary antibodies goat anti-rabbit/mouse (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at RT proteins were visualized by enhanced chemiluminescence (BioRad). For loading control, a Coomassie-stained SDS-polyacrylamide gel was used for whole protein in the supernatant and GAPDH was used for the whole cell lysate.
Gelatin is a substrate of MMP2 and can be used for detection of activity in supernatant. Equal amounts of supernatant (4 μg) used for Western blot were applied under non-reducing conditions on 10% copolymerized gelatin-polyacrylamide gels and electrophoresed (BioRad) in running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS). Gels were washed 2 times in 2.5% Triton-X-100/ddH2O for 15 min following incubation in development buffer (50 mM Tris, 5 mM CaCl2, 0.02% NaN3) for 4 h. Gels were fixed by shaking in methanol:ethanol:acetic-acid (4.5 : 4.5 : 1) for 15 min and stained with fixation buffer containing 0.1% Coomassie for 2 h. Since gelatin is a protein, the whole gel is stained by Coomassie. Gels were incubated in fixation buffer until transparent bands appeared. The more gelatin is cleaved by MMP2, the more non-stained area is visible. Gels were visualized by a standard desktop scanner. Coomassie staining of total protein in conditioned media was used to demonstrate that equal numbers of cells were used during the conditioning of the media.
Nuclear translocation of NFκB
For nuclear translocation of NFκB, 10 000 cells were cultured on 12 mm coverslips in complete medium. Cells were treated with 5 μM JS-K for 48 h or 10 ng/ml TNFα for 6 h. Untreated cells were used as controls. Cells were fixed for 10 min with 4% PFA at RT and permeabilized for 15 min in 0.5% PBST at 4 °C. Coverslips were incubated with 2% bovine serum albumin/5% normal goat serum/PBS blocking solution for 30 min at 37 °C and 30 min at RT before 2 h incubation of α NFκB p65 antibody (1 : 200, sc-8008 Santa Cruz, CA, USA) at RT in blocking solution. After washing with 0.05% PBST, cells were incubated with secondary goat α mouse antibody (Alexa 568, 1 : 5000, Invitrogen, Thermo Fisher, Bonn Germany) for 1 h at RT. Nuclei were counterstained with DAPI (10 ng/ml, Sigma-Aldrich, Steinheim, Germany) for 5 min and mounted with FluoromountG (Dako, Hamburg, Germany). Fluorescence was visualized by microscopy (×20, Zeiss Axio Observer). Scale bars represent 200 μm.
All experiments were performed in triplicates. Data are shown as mean±S.D. Data were compared using an unpaired two-tailed student’s t-test; P<0.05 was considered statistically significant.