Heme oxygenase-1 in macrophages controls prostate cancer progression.

Innate immune cells strongly influence cancer growth and progression via multiple mechanisms including regulation of epithelial to mesenchymal transition (EMT). In this study, we investigated whether expression of the metabolic gene, heme oxygenase-1 (HO-1) in tumor microenvironment imparts significant effects on prostate cancer progression.We showed that HO-1 is expressed in MARCO-positive macrophages in prostate cancer (PCa) xenografts and human prostate cancers. We demonstrated that macrophage specific (LyzM-Cre) conditional deletion of HO-1 suppressed growth of PC3 xenografts in vivo and delayed progression of prostate intraepithelial neoplasia (PIN) in TRAMP mice. However, initiation and progression of cancer xenografts in the presence of macrophages lacking HO-1 resulted in loss of E-cadherin, a known marker of poor prognosis as well as EMT. Application of CO, a product of HO-1 catalysis, increased levels of E-cadherin in the adherens junctions between cancer cells. We further showed that HO-1-driven expression of E-cadherin in cancer cells cultured in the presence of macrophages is dependent on mitochondrial activity of cancer cells.In summary, these data suggest that HO-1-derived CO from tumor-associated macrophages influences, in part, E-cadherin expression and thus tumor initiation and progression.


INTRODUCTION
The tumor microenvironment provides unique conditions to regulate cellular transformation and cancer growth [1,2]. Understanding the balance of signals produced by stromal immune cells in the tumor microenvironment facilitates new targeted therapies for cancer treatment [3,4]. Tumor-associated macrophages (TAMs) and infiltrating myeloid cells constitute the main components of the tumor stroma and are commonly designated as M2 polarized or alternatively activated macrophages (AAM) [1,2,5].
Epithelial to mesenchymal transition (EMT) is characterized by loss of E-cadherin and thus loss of cell adhesion, gain of migratory capacity, and increased invasiveness, stemness, and resistance to apoptosis [6,7]. www.impactjournals.com/oncotarget EMT generally occurs at the invasive front of metastatic tumors where TAMs accumulate [8] and has been linked to high expression of Twist and Snail [9], changes in mitochondrial activity, reactive oxygen species (ROS) production, and modulation of glucose and lipid metabolism [10,11]. Knockdown of mitochondrial complex I increased ROS production and led to enhanced migration, invasion, and spheroid formation [12]. Similarly, a mutation in the NADH dehydrogenase subunit 6 generates a deficiency in respiratory complex I, leading to ROS overproduction and enhanced metastatic potential of cancer cells [13]. We recently showed that carbon monoxide (CO) requires functional mitochondria to induce cell cycle arrest and apoptosis of prostate cancer xenografts [14,15]. However, the link between regulation of cancer metabolism and specifically how heme degradation pathways influence tumor-associated macrophages and EMT in cancer cells remains poorly defined.
Heme oxygenase-1 (HO-1) is a metabolic protein responsible for the degradation of heme to carbon monoxide (CO), bilirubin, and iron [16]. Although the role of HO-1 in macrophages in models of sepsis and injury is well-recognized, HO-1 expression in the tumor microenvironment remains to be investigated [17]. We have recently shown that HO-1 is a critical regulator of myeloid cell differentiation of CD14 high macrophages [18]. Others and we have shown that HO-1 is present in the nucleus of moderately differentiated tumors and is associated with tumor progression [19][20][21]. HO-1 induction favors epithelial phenotype by preventing the loss of E-cadherin and increased α-smooth muscle actin expression in rat renal fibrosis [22]. In line with this observation, treatment with heme fostered an E-cadherin/ β-catenin interaction at cell-cell junctions and high induction of HO-1 by heme was associated with increased levels of E-cadherin [23]. Further, beneficial effects of HO-1 on reversing EMT were demonstrated in human peritoneal mesothelial cells [24].
In this study, we evaluated the role of HO-1 in myeloid cells in regulation of tumor growth and progression. Further, we hypothesized that HO-1 controls coupling of metabolism and respiration between cancer cells and macrophages. We demonstrated a role for HO-1 in immune cell in tumor progression via regulation of mitochondrial activity and expression of E-cadherin in tumor cells.

HO-1 is expressed in tumor-associated macrophages
Others and we have previously demonstrated that HO-1 is highly expressed in prostate cancer [14,15] and that HO-1 in cancer cells is targeted to the nucleus and remains enzymatically inactive to drive a malignant cell phenotype [25]. Overexpression of enzymatically active HO-1 in A549 lung carcinoma cells blocks tumor xenograft growth as compared to xenografts established from A549 cells stably transfected with control empty vector (control vector: 406 ± 58 mm 3 , HO-1: 134 ± 28.4 mm 3 , p < 0.05). However, because macrophages expressed high levels of HO-1, we reasoned that HO-1 in myeloid cells in the tumor microenvironment may also influence tumor growth and spread. We found that in addition to nuclear HO-1 in cancer cells in tumor xenografts, HO-1 is also expressed in stromal cells, specifically in the cytoplasm of MARCO positive macrophages in tumors ( Figure 1A). HO-1 was not present in F4.80 high or MMR high macrophages in tumor stroma (data not shown).
Since immune cells are important for the invasion and metastatic spread of tumors, we first correlated HO-1 expression with macrophage markers. We evaluated HO-1 mRNA expression in available GEO profiles from 18 normal prostate tissues, 62 samples adjacent to tumor tissue, 64 primary prostate tumors and 24 prostate cancer metastases [26]. We found significantly higher expression of HO-1 in metastatic prostate cancer samples as compared to normal tissues and primary tumor samples ( Figure 1B). Similarly, we showed increased expression of TAM markers such as MARCO and CCR2 in metastatic biopsies as compared to primary cancer specimens ( Figure 1C-1D). We demonstrated a significant and positive correlation between HO-1 and MARCO expressions in all samples (Table 1).
We have further evaluated HO-1 protein expression in human prostate cancer and BPH biopsies, which allowed us to gain information about HO-1 levels and location in tumor and stroma cells. We confirmed that HO-1 is expressed in both cancer and stromal cells as well as in infiltrating immune cells ( Figure 1E-1F). Interestingly, we found that HO-1 expression in the nuclei of tumor cells was significantly higher in cancer versus benign prostatic hyperplasia (BPH), while lower expression of HO-1 was detected in stromal cells as compare to nuclear staining in PCa ( Figure 1F). However, only few samples in our cohort showed infiltration of leukocytes and therefore we were unable to correlate the levels of HO-1 in immune cells with clinical parameters.
These data suggest that changes in the expression of HO-1 both in the nucleus as well as in stroma might influence cancer progression.

TAM-derived HO-1 expressed in differentiated macrophages modulates tumor initiation and progression
To investigate the role of HO-1 in tumor stroma with focus on TAMs during tumor development and progression we have used the well-established model of prostate cancer in TRAMP mice [15], which were crossed to knockout mice with specific deletion of HO-1 in myeloid cells (Figure 2A   To directly test a role of differentiated macrophage-derived HO-1 in regulation of tumor development, we inoculated PC3 cells with bone marrow derived macrophages (BMDM) expressing HO-1 (PC3+Hmox1 flfl ) or lacking HO-1 (PC3+LyzM-Cre:Hmox1 flfl ) into the flanks of nude mice. Surprisingly, the presence of differentiated macrophages in PC3 xenografts resulted in significantly slower growth of tumors as compared to PC3 cells derived-xenografts ( Figure 2C). Deletion of HO-1 in myeloid cells (LyzM-Cre-Hmox1 flfl ) further significantly suppressed tumor growth as shown by Ki67 staining and growth curve evaluation ( Figure 2C-2D, #p < 0.05, PC3+Hmox1 flfl versus PC3+LyzM-Cre:Hmox1 flfl ). These data together with the direct deletion of HO-1 in myeloid cells in TRAMP model suggest that HO-1 in macrophages is a positive regulator of tumor growth or transformation due to induction of M2 polarization [27][28][29], change in macrophage differentiation [18] or influence on other immune cells in tumor microenvironment.
In vitro co-culture of PC3 cell with macrophages expressing HO-1 (Hmox1 flfl) ) or lacking HO-1 (LyzM-Cre-Hmox1 flfl ) demonstrated that HO-1 expressing macrophages significantly suppressed tumor growth after 24 h compared to generate myeloid linage specific knockout of HO-1 (LyzM-Cre:Hmox1 fl/fl ), which were further crossed to TRAMP mice. Tumor initiation was measured at 25 weeks of age by evaluation of Ki67 positivity within the prostate glands. Sections were also stained with E-cadherin, loss of which is a marker of tumor progression. Number of glands per field of view (FOV) with prostatic intraepithelial neoplasia (PIN) or adenocarcinoma lesions were evaluated as incidence of tumorigenesis based on H&E staining in n = 5-6 mice per group. Data ± SD are shown, *p < 0.05 (one-tailed t-test). C-D. PC3 cells or PC3 cells mixed with BMDM from Hmox1 fl/fl and LyzM-Cre:Hmox1 fl/fl mice were inoculated into the flanks of nude mice. Xenograft growth was measured over time (C) and Ki67 staining (D) was performed at day 26 after inoculation. *p < 0.01, PC3 versus PC3+ BMDM from Hmox1fl/fl mice and #p < 0.05, PC3+ BMDM from Hmox1 fl/fl versus PC3+ BMDM from LyzM-Cre:Hmox1 fl/fl ; n = 3-5 mice per group. E. BrdU incorporation assay was employed to measure proliferation of PC3 co-cultured with BMDM from LyzM-Cre:Hmox1 fl/fl and Hmox fl/fl mice in the ratio of 1:2 or 1:5 (PC3:BMDM) for 24 h. Data are representative for 3 independent experiments in triplicates. *p < 0.05. www.impactjournals.com/oncotarget to PC3 control, however macrophages lacking HO-1 failed to significantly alter growth of PC3 cells ( Figure 2E). This suggests that presence or infiltration of other cell types in the tumor microenvironment may dictate the suppressive effect of macrophages lacking HO-1 in vivo.

HO-1 in macrophages controls immune cell infiltration to the tumor microenvironment
Since cancer growth is controlled by multiple cell types in the tumor microenvironment, we tested whether HO-1 in macrophages could influence infiltration of immune cells and thus affect tumor development. In contrast, in PC3 xenografts injected with myeloid cells with deletion of HO-1, the number of NK and Ki67 positive cells did not increase as compared to PC3 xenograft alone ( Figure 3E-3F). However, there was a significant inhibition in number of Gr-1 positive cells in xenografts with the absence of HO-1 in macrophages suggesting the specificity of HO-1 in controlling NK cell infiltration (Figure 3).

TAMs control EMT in vivo
Since the control of tumor growth by macrophages was unexpected, we next asked whether differentiated myeloid cells with high HO-1 expression influences EMT. Co-inoculation of macrophages with cancer cells allow for re-expression of E-cadherin, a marker of less aggressive epithelial cancer phenotype. Interestingly, lack of HO-1 in macrophages resulted in decreased expression of E-cadherin ( Figure 4A) and increased mesenchymal markers Twist-1 and Snail as compared to macrophages with high HO-1 expression ( Figure 4A-4C). In contrast, overexpression of full length or truncated (partially located in the nucleus) HO-1 in PC3 cells did not significantly altered expression of E-cadherin ( Figure 4D-4E). This suggests that lack of HO-1 in myeloid cells rather than in cancer cells in the co-culture model of differentiated macrophages and cancer cells may slow tumor growth while leading to more aggressive and invasive cancer. Inverse correlation between cancer growth and invasive phenotype has been previously described [30,31].

HO-1 derived CO targets STAT3 and mitochondrial pathways to control EMT
Since STAT3 was shown to mediate the effects of HO-1-derived and exogenous CO [32], we next looked at the effects of HO-1 in tumor microenvironment on STAT3 signaling. We found that lack of HO-1 in myeloid cells resulted in lower activation of STAT3 in the co-culture of PC3 and BMDM in vivo and in vitro as measured by decreased levels of phosphorylated STAT3 ( Figure 4A, 4B, 4D, 4F).
The majority of the effects of HO-1 are mediated by the generation of one or more heme degradation products. Therefore, we also tested whether CO influenced macrophage-mediated effects on PC3 cells ( Figure 4G & 5). Treatment with CO for 24-48 h resulted in higher expression of E-cadherin as compared to air treated PC3 cells ( Figure 4G). Further, exposure of PC3 to CO at 250 ppm for 24 h resulted in suppression of cancer cell growth ( Figure 5A). Similarly, BMDM expressing HO-1 blocked cancer cell growth, which was further inhibited by CO ( Figure 5A-5C). We previously showed that CO suppresses growth of human xenografts as well as TRAMP and KRAS-driven tumors in mice [15]. We next tested whether HO-1-derived CO and exogenous CO modulates macrophage phenotype when co-cultured with cancer cells. Lack of HO-1 leads to lower arginase (M2 marker) and iNOS (M1 marker) expression as compared to wild type BMDM ( Figure 5B) suggesting the role of HO-1 in maturation and polarization of macrophages [18]. Both markers were further suppressed by application of exogenous CO ( Figure 5B). Evaluation of RANTES and mannose receptor (MMR) mRNA levels in macrophages cocultured with PC3 cells showed higher levels of RANTES and lower mannose receptor (MMR), indicating M1 macrophage polarization in the presence of PC3 cancer cells, and that this polarization was in part augmented by CO ( Figure 5C-5D). These data suggest that inefficient macrophage polarization in the absence of HO-1 or CO may be a contributing factor to the effects on cancer growth and EMT.
In efforts to understand how macrophages were influencing EMT, we tested the role of mitochondrial respiration, which is a target for HO-1-derived CO or exogenous CO. We used Mitotracker probe that labels active mitochondria ( Figure 6A-6B) and showed that mitochondrial staining was much stronger under the cell membrane in PC3 cells co-cultured with BMDM as compared to PC3 alone ( Figure 6A-6B). Further, presence of macrophages increased expression of E-cadherin on the cell surface. To evaluate whether differential polarization of macrophages influenced E-cadherin expression, we co-cultured M1-like or M2like macrophages with PC3 cells. We showed that M2like macrophages strongly induced E-cadherin expression on PC3 cells, while M1-like macrophages only slightly upregulated E-cadherin on the surface of cancer cells ( Figure 6C).
We reasoned that there might be a correlation between higher mitochondria activity and E-cadherin assembly in PC3 cells in the presence of macrophages. We found that the presence of macrophages resulted in higher internal respiratory capacity in cancer cells in response to the mitochondrial stressor FCCP as compared to PC3 cells or macrophages cultured alone ( Figure 6D). Indeed the presence of macrophages in the co-culture experiments resulted in restoration of expression of the mitochondrial transcription factor A (mTFA) and grp-78 that were otherwise suppressed after treatment with FCCP ( Figure 6E).

Negative correlation between HO-1 and E-cadherin in patient samples
To evaluate the clinical significance of our studies on the role of HO-1 in regulation of EMT, we compared expression of HO-1 and E-cadherin in primary BPH and prostate tumors. We show a strong positive correlation between expression of cytoplasmic HO-1 and E-cadherin ( Figure 7A-7B). There was no significant correlation between nuclear tumor-derived HO-1 or stromal HO-1 with expression of E-cadherin ( Figure 7C-7D). However, lower stromal expression of HO-1 in tumor biopsies as compared to BPH may indicate metastatic switch, which leads to loss of E-cadherin in primary tumors (Figure 1). High nuclear expression of HO-1 in cancer cells may be an independent driver of invasion [33] while cytoplasmic HO-1 or myeloid cells-derived HO-1/CO may suppress EMT and cancer progression [15,34].

DISCUSSION
Immune cells in the tumor microenvironment shape multiple properties of cancer cells including their invasive phenotype, proliferation and survival. In this study, we asked whether and how the metabolic enzyme HO-1 influences the TAM phenotype and thus influences cancer cell growth and metastasis.
In this study, we identified HO-1 in myeloid cells to be an important regulator of tumor progression and EMT. We show that cell-specific deletion of HO-1 in myeloid cells suppressed tumor initiation and growth while increasing EMT in different models of prostate cancer. Furthermore, we identified mitochondrial respiration in macrophages as a modulator of EMT and E-cadherin expression.
We found that HO-1 was expressed in MARCO positive macrophages in established tumors and that there was a strong correlation between both HO-1 and MARCO expression in prostate cancer metastases.
MARCO is a class I scavenger receptor that mediates antibody-dependent phagocytosis of pathogen as well as of cancer cells and is expressed in M2 macrophages [35]. Presence of HO-1 in MARCO-positive macrophages may indicate the mechanism for regulation of tumor growth via enhanced polarization of macrophages towards a proangiogenic M2 phenotype. Expression of HO-1 did not correlate with levels of CCR2, a macrophage receptor, which allows for monocyte recruitment into tumors and metastatic spread of breast cancer in mice models as well as marker of M1 polarization [36]. Indeed, cytoplasmic HO-1 correlated with higher level of E-cadherin, accordingly to the studies by Gueron et al. showing that heme induced E-cadherin and change in cell morphology in prostate cancer cells [23].
Polarization of macrophages towards an M1 or M2 phenotype dictates the immune response of T cells as well as angiogenesis and metastatic growth. We found that the presence of mature macrophages in the microenvironment at time of tumor initiation delays Expressions of HO-1 and E-cadherin were evaluated in consecutive sections of 35 prostate cancer and corresponding BPH samples. Cytoplasmic, nuclear and stromal HO-1 were evaluated and correlated with E-cadherin expression in the samples. Note: Cytoplasmic but not nuclear or stromal HO-1 correlates with E-cadherin expression. growth of spontaneous tumors, syngeneic transplants or xenografts. Number of differentiated macrophages in the tumor microenvironment might be a limiting factor that allows for dormancy of tumors and suppression of development of advanced and metastatic disease. Depletion of HO-1 from differentiated TAMs (in xenograft model) further suppressed the growth of cancer cells but initiated EMT. Indeed, HO-1 in TAMs has been associated with accelerated tumor growth in breast cancer [37]. HO-1 is a strong inhibitor of acute inflammation and therefore may block inflammation in cancer [38]. Further, HO-1 regulates myeloid differentiation [18] as well as immune response to viral [39] or bacterial infection via increased IL-1β release [40]. HO-1 in the tumor niche promoted lung metastases by controlling VEGF and IL-10 production [41]. The balance between proinflammatory signaling and anti-inflammatory mediators is often disrupted in solid tumors and is a hallmark of carcinogenesis [4].
Cancer cells blocked in the G1 cell cycle phase migrate and invade more than cells that undergo mitosis [30]. The presence of immune cells such as macrophages with a specific polarization phenotype can differentially modulate the growth and invasive phenotype of cancer cells. Our xenograft data suggest that presence of terminally differentiated macrophages lacking HO-1 suppresses tumor growth and induces E-cadherin expression. However, we did not find similar effect on EMT in TRAMP mice, where expression of E-cadherin correlated directly with Ki67 and tumor progression, and was higher in PIN lesions in TRAMP mice lacking HO-1 in myeloid cells. We speculate that a difference might be due to lack of T cells in the nude mice (xenografts) and therefore possible change in the infiltration of immune cells into cancer microenvironment. Further, the number of TAMs infiltrating xenografts as well as TRAMP tumors might be different. We injected terminally differentiated macrophages with cancer cells in xenograft model, in which we showed a direct influence of macrophages on cancer growth and EMT. In TRAMP mice, myeloid cells are incorporated during tumor growth and their phenotype and regulatory functions might be altered by cancer cells. These myeloid cells can be skewed to myeloid suppressor cells or TAMs, which support tumor growth. Characterization of the role of HO-1 in such conditions remains to be investigated in future study.
Increased expression of HO-1 restricts differentiation and polarization of TAMs which in turn regulate cancer cell invasion and growth. Myeloid HO-1 is known to drive macrophage differentiation [18]. Further, HO-1 knockout mice have decreased IRF3 function with defects in early immune responses [39]. We showed that HO-1 in macrophages is likely an important source of CO, which diffuses to the environment and targets E-cadherin in neighboring cancer cells. The role of HO-1 in modulating E-cadherin expression has been addressed in PC3 using heme treatment in a work by Gueron et al [1]. Overall heme treatment induced E-cadherin expression in cancer cells [1]. Overexpression of a truncated HO-1 (t-HO-1) lacking the TMS in HeLa and H1299 cells promoted cell proliferation and migration/invasion [2]. We showed that overexpression of either cytoplasmic or nuclear HO-1 in PC3 cells did not have significantly altered E-cadherin or Snail expressions ( Figure 4D-4E). We speculate that a balance of cytoplasmic and nuclear HO-1 in cancer cells and myeloid cells derived HO-1 may dictate changes in E-cadherin expression.
E-cadherin plays a critical role in the function of the epithelial layer and is lost during EMT. E-cadherins are also recognized by Killer cell lectin-like receptor subfamily G member 1 (KLRG1) receptor on NK cells [42,43]. NK cells in turn not only directly kill cancer cells or cells infected with virus but also generate cytokines such as interferon-γ which induces production of proinflammatory cytokines in the tumor stroma. INFγ is critical for M1 polarization of macrophages that inhibit tumor growth [44]. Alternatively activated M2 polarized TAM promote EMT in pancreatic cancers via activation of TLR-IL-10 pathway [45]. Lack of HO-1 correlates with less mature and polarized macrophages in tumor microenvironment, which decreases the number of NK cells being recruited to the tumor.
We observed a significant negative correlation of HO-1 in the cytoplasm and E-cadherin in cancer cells of specimens from prostate cancer patients. Presence of HO-1-positive macrophages in the tumor microenvironment might be an indicator of expression of E-cadherin and other EMT markers. HO-1 is a metabolic gene that controls energy balance via targeting of mitochondria through generation of CO [16,38]. Assembly of E-cadherin based junctions requires energy and therefore the link between increased mitochondrial content in cancer cells and less aggressiveness of tumors may be important for therapeutic targeting. Depletion of ATP results in internalization of almost 70% of the E-cadherin from the cell surface [46] suggesting that energy from glucose metabolism is required for E-cadherin junctions. We showed that increased mitochondria activity in cancer cells via presence of macrophages leads to significantly increased E-cadherin expression. The presence of macrophages in co-culture with cancer cells leads to significant restoration of the spare respiratory capacity in cancer cells as compared to in culture of cancer cells alone. Dysfunction of the tricarboxylic acid (TCA) cycle induces tumor growth due to activation of a pseudo-hypoxic pathway under normoxic conditions [47,48].
In summary, we showed that HO-1 in TAMs dictates cancer growth and metastases and as such can be a target for modulation of immune cell function during tumor dormancy and progression. www.impactjournals.com/oncotarget 36 human primary prostate cancers (PC) and 24 benign prostatic hyperplasia (BPH) samples were obtained from the cancer bank from the Department of Pathology at Lund University in Malmo, Sweden. Prostate cancer samples and BPH tissues from patients with Gleason grade 3-4 tumors and BPH samples were selected by certified pathologist (Dr. Martin Johansson, Lund University) for the tissue microarray (TMA) and printed in a TMA format in duplicates. Investigation has been conducted in accordance with the ethical standard and according to the Declaration of Helsinki and according to national and international guidelines and has been approved by the authors' institutional review board.

Geo data
GEO profiles from 18 normal prostatic tissues (without any pathological alterations), 62 tissues adjacent to tumors, 64 primary tumors and 24 metastatic samples were obtained from patients with prostate cancer as described in previously published data [26]. Specifically, 24 metastatic biopsies were derived from 4 patients with prostate cancer metastases to the liver, lymph nodes, kidney, lung and adrenal glands [26].
PC3 cells were co-cultured with BMDM from Hmox1 fl/fl or LyzM-Cre:Hmox1 fl/fl mice in a 1:2 or 1:5 ratio for 24 h. PC3/BMDM cells alone or in co-culture were used for immunostaining using anti-E-cadherin antibody (CatNo: ab15148) and Mitotracker Red CMXRos (Life Technologies), for detecting mitochondria as well as for direct testing of mitochondrial activity Mitochondria Stress Kit (Seahorse) was used as previously described [49].
Overexpression of HO-1: Constructs were kindly provided by Dr. Chau (Taiwan) and forced overexpressions of Flag, HO-1-Flag or truncated HO-1-Flag (t-HO-1) were established in PC3 cells upon transfection with Amaxa. Forty eight hours after transfection, cells were stained or cell lysates were immunoblotted.

Imunohistochemistry and immunostaining
Paraffin embedded formalin or zinc fixed tissues were processed for staining as previously described [15]. The following antibodies were applied: HO-1 (Abcam), MARCO (BD Biosciences), P-Stat3 (Cell Signaling), E-cadherin (Cell Signaling), F4.80 (BD Biosciences), Ki67 (Dako, Clone TEC3, rat monoclonal anti-mouse Ki67) and Gr1 (BD Biosciences). Briefly, slides were incubated with primary antibodies overnight at 4°C. After washing with PBS, slides with tissues were blocked with H 2 O 2 followed by incubation with biotin-labeled secondary antibodies for 1 h at RT. VECTASTAIN Elite ABC System was used to enhance the signals (Vector Laboratories). DAB substrate (Vector Laboratories) was used for developing the reactions. After mounting the slides they were covered and analyzed by light microscopy.
Immunofluoresence staining was carried out as previously described [15]. Briefly, 2% paraformaldehyde fixation with subsequent washing, blocking and staining with primary and fluorescently labeled secondary antibody was applied (Alexa Fluor488 or Alexa Fluor594; Life Technologies, NY, USA). Nuclei were stained with Hoechst and immunofluorescence staining was analyzed by Zeiss Apotome fluorescence microscope.