Conceived and designed the experiments: AB TA LGK TAZ. Performed the experiments: AB TA NK LGK TAZ. Analyzed the data: AB TA NK LGK TAZ. Contributed reagents/materials/analysis tools: DCG SK. Wrote the paper: AB TA TAZ.
Current address: Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America
The authors have declared that no competing interests exist.
Cachexia, or weight loss despite adequate nutrition, significantly impairs quality of life and response to therapy in cancer patients. In cancer patients, skeletal muscle wasting, weight loss and mortality are all positively associated with increased serum cytokines, particularly Interleukin-6 (IL-6), and the presence of the acute phase response. Acute phase proteins, including fibrinogen and serum amyloid A (SAA) are synthesized by hepatocytes in response to IL-6 as part of the innate immune response. To gain insight into the relationships among these observations, we studied mice with moderate and severe Colon-26 (C26)-carcinoma cachexia.
Moderate and severe C26 cachexia was associated with high serum IL-6 and IL-6 family cytokines and highly similar patterns of skeletal muscle gene expression. The top canonical pathways up-regulated in both were the complement/coagulation cascade, proteasome, MAPK signaling, and the IL-6 and STAT3 pathways. Cachexia was associated with increased muscle pY705-STAT3 and increased STAT3 localization in myonuclei. STAT3 target genes, including SOCS3 mRNA and acute phase response proteins, were highly induced in cachectic muscle. IL-6 treatment and STAT3 activation both also induced fibrinogen in cultured C2C12 myotubes. Quantitation of muscle versus liver fibrinogen and SAA protein levels indicates that muscle contributes a large fraction of serum acute phase proteins in cancer.
These results suggest that the STAT3 transcriptome is a major mechanism for wasting in cancer. Through IL-6/STAT3 activation, skeletal muscle is induced to synthesize acute phase proteins, thus establishing a molecular link between the observations of high IL-6, increased acute phase response proteins and muscle wasting in cancer. These results suggest a mechanism by which STAT3 might causally influence muscle wasting by altering the profile of genes expressed and translated in muscle such that amino acids liberated by increased proteolysis in cachexia are synthesized into acute phase proteins and exported into the blood.
Cachexia, or progressive wasting of fat and skeletal muscle despite adequate nutrition, is a pervasive and devastating complication of cancer
Clinically, cancer cachexia is defined as weight loss of at least 5% in the presence of underlying illness with associated muscle weakness, fatigue, anorexia, low lean body mass and abnormal biochemistry, including increased inflammation, anemia and low serum albumin. Weight loss of 5%, 10% or 15% total body weight is referred to as mild, moderate or severe cachexia, respectively, and both weight loss and the rate of weight loss correlate positively with mortality
The systemic metabolic derangements noted in cancer cachexia are also observed with other forms of systemic inflammation
IL-6 acts on cells by binding the IL-6 receptor α-chain (IL-6Rα), also known as gp80, either in its membrane-bound or soluble form, inducing dimerization of gp130 and activation of its associated Janus kinases (JAKs), which tyrosine phosphorylates gp130
Tisdale and Fearon with co-workers have linked cancer cachexia with persistent elevations in acute phase response proteins in serum
We sought to determine potential relationships between elevated cytokines, the acute phase response and wasting in cancer. Here we report characterization of the serum cytokines and the muscle transcriptome in the colon-26 adenocarcinoma model of cancer cachexia. We provide evidence of STAT3 activation, target gene expression and the acute phase response in both liver and skeletal muscle in these mice, providing a molecular link between these observed phenomena.
Implantation of colon-26 (C26) adenocarcinoma cells into Balb/c or CD2F1 mice is a classic model of cancer cachexia
A, B, Body weight changes for control mice and for mice injected with C26 tumor cells. Mice euthanized on day 19 had lost ∼10% body weight, considered moderate cachexia. Mice euthanized on day 24 had lost ∼15% body weight, considered severe cachexia. C, Significantly decreased quadriceps and gastrocnemius weights were observed with C26 cachexia. Differences were not significant in muscles from moderate versus severe cachexia. D, Liver weight and tumor weight both increased with severity/duration of cachexia. E, Representative Western blotting for Myosin Heavy Chain (MyHC) in quadriceps muscle from both control and tumor bearing mice. F, Densitometric quantification of the Western blotting for MyHC protein shows a marked reduction in both moderate (−69% vs. controls) and severe (−81% vs. controls) cachexia. G, Total protein content in quadriceps muscle from control and tumor-bearing mice. Protein content is significantly reduced in both moderate (−7% vs. controls) and severe (−13% vs. controls). n = 4–5 per group; **P<0.01, ***P<0.001.
In contrast to muscle wasting, total liver mass increased 17.3% (
Plasma analyte profiling of both moderately and severely cachectic mice revealed that circulating levels of IL-6 and three other gp130 ligands, IL-11, leukemia inhibitory factor (LIF) and Oncostatin M were significantly increased in tumor-bearing mice (
Normal | Moderate Cachexia | Severe Cachexia | |
|
|||
IL-6 (pg/ml) | <LOW> | 104±41 |
120±22 |
IL-11 (pg/ml) | <LOW> | 2010±122 |
3180±1424 |
LIF (pg/ml) | 1570±170 | 1997±165 |
2137±172 |
OSM (ng/ml) | <LOW> | 0.121±0.060 |
0.220±0.024 |
|
|||
TNF (ng/ml) | <LOW> | 0.063±0.006 |
0.049±0.005 |
IL-1α (pg/ml) | 62±17 | 204±90 | 214±0 |
IFN-γ (pg/ml) | <LOW> | 29±7.7 |
15±8 |
|
|||
Fibrinogen (µg/ml) | 30,800±6,350 | 165,333±21,455 |
159,667±58,586 |
Haptoglobin (µg/ml) | 17±1.3 | 164±11.5 |
173±20.1 |
Von Willebrand factor (ng/ml) | 75±36.7 | 322±49.7 |
359±65.6 |
*P<0.05.
**P<0.01.
***P<0.001 versus normal. No differences between moderate and severe cachexia were statistically significant. <LOW> indicates below the level of detection.
We sought to identify changes in muscle gene expression associated with wasting. Microarray analysis and data normalization was performed on RNA isolated from the quadriceps of mice with moderate or severe C26 cachexia and non-tumor bearing controls. Unsupervised hierarchical classification of the samples clearly distinguished control muscle, moderately wasted and severely wasted samples (
A, Heat map of quadriceps gene expression in C26 cachexia showing distinct clustering of genes by experimental groups. Samples are normalized to the controls. Blue indicates down-regulated genes, yellow up-regulated genes, and black no change. Only genes with
Statistical analysis was performed to identify genes exhibiting significant changes in expression during cachexia. Among 45,281 transcripts, 1607 genes were found to be differentially expressed in moderate cachexia and 1328 in severe wasting (–2≤ fold change ≥2,
Gene | Gene Description | p-value | Fold Change |
Fga | fibrinogen alpha chain | 0.0007 | 61.0 |
Itih3 | inter-alpha trypsin inhibitor, heavy chain 3 | 0.0003 | 59.2 |
Otop1 | otopetrin 1 | 5.50E-05 | −59.2 |
Egln3 | EGL nine homolog 3 | 7.40E-05 | 55.6 |
Ucp1 | uncoupling protein 1 (mitochondrial, proton carrier) | 0.0176 | −44.1 |
Anxa13 | annexin A13 | 0.0158 | 34.8 |
Pdzd7 | PDZ domain containing 7 | 0.002 | −34.7 |
Trim7 | tripartite motif-containing 7 | 0.0017 | −31.3 |
Saa1 | serum amyloid A 1 | 0.008 | 29.9 |
Cxxc6 | tet oncogene 1 | 0.0243 | −29.6 |
Ntrk3 | neurotrophic tyrosine kinase, receptor, type 3 | 0.001 | −29.6 |
Il1r2 | interleukin 1 receptor, type II | 0.0068 | 26.6 |
Doc2b | double C2, beta | 0.0075 | 25.6 |
Snf1lk | salt inducible kinase 1 | 0.001 | 25.4 |
Apoa1 | apolipoprotein A–I | 0.0124 | 24.5 |
Mmd2 | monocyte to macrophage differentiation-associated 2 | 0.0059 | −24.4 |
Aldh1a7 | aldehyde dehydrogenase family 1, subfamily A7 | 0.0044 | −24.4 |
Ambp | alpha 1 microglobulin/bikunin | 0.0239 | 22.1 |
Aqp4 | aquaporin 4 | 0.0352 | −21.1 |
Scgb3a1 | secretoglobin, family 3A, member 1 | 0.0014 | 21.1 |
Dynlrb1 | dynein light chain roadblock-type 1 | 6.40E-05 | −20.6 |
Lypd6 | LY6/PLAUR domain containing 6 | 0.0365 | −18.9 |
Cxcl13 | chemokine (C-X-C motif) ligand 13 | 0.0162 | 18.6 |
Tmem118 | ring finger protein, transmembrane 2 | 0.0369 | −18.5 |
Scg3 | secretogranin III | 6.20E-05 | −18.3 |
Plcd4 | phospholipase C, delta 4 | 0.0284 | −17.9 |
Lrrc38 | leucine rich repeat containing 38 | 0.016 | −17.3 |
Maff | v-maf musculoaponeurotic fibrosarcoma oncogene family, protein F | 0.0068 | 17.2 |
Hpx | hemopexin | 0.0018 | 17.1 |
Gene | Gene Description | p-value | Fold Change |
Nnat | neuronatin | 1.40E-05 | −96.2 |
Cilp2 | cartilage intermediate layer protein 2 | 0.0002 | −39.4 |
Lcn2 | lipocalin 2 | 0.007 | 33.1 |
Saa1 | serum amyloid A 1 | 0.0107 | 33.0 |
Gm1611 | gene model 1611, (NCBI) | 0.0015 | −32.9 |
Itih3 | inter-alpha trypsin inhibitor, heavy chain 3 | 0.0004 | 29.9 |
Scgb3a1 | secretoglobin, family 3A, member 1 | 0.0007 | 29.1 |
Rsph1 | radial spoke head 1 homolog (Chlamydomonas) | 1.50E-09 | −28.4 |
Cxcl13 | chemokine (C-X-C motif) ligand 13 | 0.0086 | 24.0 |
Lypd6 | LY6/PLAUR domain containing 6 | 0.0044 | −22.2 |
Aqp4 | aquaporin 4 | 0.0024 | −21.1 |
Myoz3 | myozenin 3 | 0.008 | −20.6 |
Plcd4 | phospholipase C, delta 4 | 0.0389 | −20.4 |
Actc1 | actin, alpha, cardiac muscle 1 | 0.0004 | −19.4 |
Kcng4 | potassium voltage-gated channel, subfamily G, member 4 | 0.0006 | −18.4 |
Rapgef6 | Rap guanine nucleotide exchange factor (GEF) 6 | 0.0099 | −18.1 |
Serpina3m | serine (or cysteine) peptidase inhibitor, clade A, member 3M | 0.0058 | 16.2 |
Csf2rb2 | colony stimulating factor 2 receptor, beta 2, low-affinity (granulocyte-macrophage) | 0.0285 | 15.7 |
D0H4S114 | DNA segment, human D4S114 | 0.0004 | −15.7 |
Itih4 | inter alpha-trypsin inhibitor, heavy chain 4 | 0.0001 | 15.7 |
Vsig4 | V-set and immunoglobulin domain containing 4 | 0.0138 | 15.45 |
Mmd2 | monocyte to macrophage differentiation-associated 2 | 0.0358 | −15.4 |
Il1r2 | interleukin 1 receptor, type II | 0.0173 | 15.2 |
Pld5 | phospholipase D family, member 5 | 0.0068 | −13.9 |
Mt2 | metallothionein 2 | 0.0184 | 13.9 |
Adamts4 | a disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 motif, 4 | 0.0003 | 13.0 |
Serpina3n | serine (or cysteine) peptidase inhibitor, clade A, member 3N | 0.0048 | 13.0 |
Gdap1 | ganglioside-induced differentiation-associated-protein 1 | 0.0181 | −12.6 |
3526401B18Rik | RIKEN cDNA 3526401B18 gene | 0.0048 | −12.6 |
Chad | chondroadherin | 0.0023 | −12.6 |
Muscle wasting of virtually all etiologies is associated with increased expression of skeletal muscle ubiquitin E3 ligases, Trim63/Murf-1 and Fbxo32/Atrogin-1
A, Expression of the ubiquitin ligases, Fbxo32/Atrogin-1 and Trim63/MuRF1 are induced by microarray and qPCR analysis. Expression of genes encoding muscle structural proteins, including ACTA1 and MHY8 are decreased. B, Gene expression patterns in C26 cachexia mapped onto a schematic of muscle growth regulation pathways. Genes with demonstrated growth-promoting activity in muscle are shown in green, growth inhibitory genes in red. The number on the left represents fold-change in moderate cachexia and right in severe cachexia, with the arrow indicating the direction of the change. Genes with no values were either not changed or not present in the dataset.
In order to identify molecular pathways regulated in response to C26-induced cachexia, gene profiles from quadriceps of control, moderate and severe cachexia were analyzed using NextBio (
A. The top 20 Broad MSigDB canonical pathways significantly up-regulated in moderate and severe cachexia versus controls. Pink bars (upper axis) represent the significance of the overlap. The triangles (moderate cachexia) and circles (severe cachexia) (lower axis) denote the number of genes differentially expressed in that pathway. Red arrows indicate pathways related to IL-6/STAT3/inflammation. B, The top 20 Broad MSigDB canonical pathways significantly down-regulated in moderate and severe cachexia versus controls. Green bars (upper axes) represent the significance of the overlap. The triangles (moderate cachexia) and circles (severe cachexia) (lower axis) as above.
The top canonical pathways down-regulated were related to skeletal muscle contraction, peroxisome proliferator-activated receptor-γ coactivator-1α, calcium regulation, extracellular matrix interactions, skeletal myogenesis, and insulin and WNT signaling, among others.
Given the prominence of the cytokine/IL-6/STAT3 pathways in the microarray analysis, we sought to characterize expression of STAT3 interacting genes in our model. Genomatix Bibliosphere was used to generate a list of the 124 documented physical and functional interactions with STAT3 in diverse systems (
A: Heat map of gene expression changes of Stat3 and co-cited gene products, as identified by Genomatix Bibliosphere. Blue indicates down regulated genes, yellow up regulated, and black no change. Only genes with P<0.05 by one-way ANOVA are shown. B: A subset of STAT3 target genes identified through the literature are differentially regulated in moderate and severe cachexia versus controls. C: STAT3 and its target genes SOCS3 and CEBPD are increased at the mRNA level by microarray and qPCR. D: Protein levels for p-STAT3 and STAT3 in protein extracts from quadriceps, gastrocnemius and liver evaluated by Western blotting analysis. E: SOCS3 protein levels. F: quantitative analysis of p-STAT3/STAT3 and p-STAT3/GAPDH ratio (expressed as fold-change vs. controls). G: quantitative analysis of SOCS3 protein levels (expressed as fold-change vs. controls). GAPDH was used as an internal reference to confirm equal loading. n = 3–5 per group; *P<0.05, **P<0.01, ***P<0.001 vs. Controls, $P<0.05 vs. moderate.
We also asked whether STAT3 target genes were also induced. Examination of the literature resulted in identification of 186 validated STAT3 target genes identified in several different species and systems (
Among the known STAT3 targets increased in quadriceps in moderate and severe cachexia were STAT3 itself and the transcription factor CCAAT/enhancer-binding protein δ (C/EBP δ)
All this robust STAT3 target gene expression suggested that STAT3 activity was increased in cachexia. STAT3 is activated in part by phosphorylation at Y705, which induces dimerization, nuclear translocation and DNA binding
A, p-STAT3 protein levels are increased in nuclear extracts prepared from quadriceps of severely cachectic C26 tumor-bearing mice compared to controls. n = 5 per group. B, Immunofluorescence analysis performed reveals increased pSTAT3 localization in myonuclei of gastrocnemius from severely cachectic C26 tumor-gearing mice (green). Nuclear staining is shown in blue (DAPI).
Given the robust induction of acute phase gene RNA in cachexia (
A. Western blotting and quantitation of fibrinogen levels in control and C26 quadriceps and liver. Data (mean ± SEM) are expressed as relative densitometry value. **P<0.01, ***P<0.001. B, Western blotting analysis of fibrinogen standard proteins and quadriceps and liver extracts for control, CHO-IL6 injected nude mice and C26 injected CD2F1 mice. Quantitation was performed on the band indicated by the arrow. Data (means ± SEM) are expressed as ng fibrinogen / µg protein. *P<0.05, **P<0.01, ***P<0.001. C, Western blotting analysis demonstrates significantly increased fibrinogen and SAA1 protein levels in quadriceps and gastrocnemius in moderate and severe C26 cachexia. *P<0.05, **P<0.01, ***P<0.001.
These results in
Despite the induction of fibrinogen and SAA mRNA in muscle, the protein levels of acute phase proteins in skeletal muscle extracts theoretically could be due to contaminating plasma. In order to further test our hypothesis that fibrinogen is produced directly from skeletal muscle following activation of the IL-6/STAT3 pathway, we infected C2C12 murine myotube cultures with a recombinant adenovirus expressing a constitutively activated form of STAT3, cSTAT3
A, Western blotting analysis and quantitation of fibrinogen in C2C12 myotubes infected with Ad-cSTAT3-GFP or Ad-GFP as control. Fibrinogen expression was increased consistent with the increase in the levels of STAT3. **P<0.01, ***P<0.001 vs. GFP. B, Western blotting analysis and quantitation of fibrinogen expression in C2C12 treated with IL-6 (100 ng/ml) for 1, 24, 48 h. GAPDH was used as loading control. Increased expression of fibrinogen was observed at each time point after IL-6 treatment. Data (means ± SEM) are expressed as relative densitometry value. ***P<0.001 vs. respective controls. C, Fibrinogen levels by ELISA of the conditioned medium of C2C12 exposed to IL-6 for 30 min, 1, 6, 24, 48 h. Fibrinogen levels were significantly elevated after 6, 24 and 48 h of IL-6 treatment. Data (means ± SEM) are expressed as ng/ml. **P<0.01, ***P<0.001 vs. controls (C).
We sought to mimic the high serum IL-6, acute phase response and muscle wasting of patients with cancer cachexia. We chose C26 adenocarcinoma, which exhibits increased circulating levels of IL-6 that coincide with muscle wasting
Regardless, IL-6 is likely not the only cytokine mediating muscle wasting in cancer or even in the C26 model. Inhibition of IL-6 only partially rescues muscle wasting in the C26 model, causing some to conclude that IL-6 is only one of several players involved in the C26 model and cannot by itself induce the full cachectic syndrome
Here we document that the STAT3 pathway is activated in skeletal muscle in C26-bearing mice and that expression of STAT3 target genes including the acute phase response genes are activated. Among the STAT3 target genes significantly induced was SOCS3, a classical feedback inhibitor of STAT3 activation. STAT3 induces expression of SOCS3 which binds to activated JAKs and receptors to inhibit STAT3 activation in at least three ways: by preventing binding of STAT to activated receptors, by binding and inhibiting activated JAKs and by targeting JAKs and receptors for degradation
Emerging data indicate that SOCS3 is regulated transcriptionally, but also post-transcriptionally and post-translationally. TNF stabilizes SOCS3 mRNA elicited by lipopolysaccharide
In this experimental work we also confirm that at least two acute phase response proteins, fibrinogen and SAA1 are expressed in muscle, and that in the case of the former, at levels about half of that expressed in the liver. The significance of these results is at least three-fold. First, they establish skeletal muscle as an important source of acute phase protein synthesis. Second, they establish a molecular link between the observations of high IL-6, increased acute phase response proteins and muscle wasting in cancer. Third, they suggest a molecular mechanism through which STAT3 might causally influence muscle wasting by altering the profile of genes expressed and mRNAs translated in muscle.
Generally, the acute phase response is considered to be hepatic in origin, although several reports document expression of acute phase response genes in lung and mammary tissue
In addition to its functional and metabolic roles, skeletal muscle is the major protein reservoir in the body. Under disease conditions, the mobilized free amino acids can also be utilized for metabolism of vital organs such as the liver, heart, brain or lung
Inflammation and correspondingly increased acute phase response protein levels are a hallmark of cancer cachexia. It has been hypothesized that hepatic synthesis of positive acute phase response proteins using amino acids liberated from skeletal muscle proteins is a major driver of skeletal muscle proteolysis, although the nature of the signal mediating both processes was not suggested
STAT3 activation has also been observed in muscle in other experimental models of cancer cachexia with high IL-6, namely ApcMin/+ mice
All animal procedures were approved by the University of Miami Institutional Animal Care and Use Committee under protocols 08–174 and 10–071. CD2F1 mice were purchased from Charles River Laboratory. Colon26 cells (a gift from Dr. Donna McCarthy) were cultured in Advanced RPMI 1640 medium supplied with 10% fetal bovine serum and 1% penicillin/streptomycin and maintained in a 5% CO2, 37oC humidified incubator. Cells were passaged when sub-confluent, and 1×106 cells per mouse were injected subcutaneously (5×105 cells in each flank). Mice were weighed daily then euthanized under isoflurane anesthesia. Tissues were collected and weighed then snap frozen in liquid nitrogen. Quadriceps and liver samples in
Murine C2C12 skeletal myoblasts (ATCC, Manassas, VA, USA) were grown in high glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, 100 mg/ml sodium pyruvate, 2 mM L-glutamine, and maintained at 37°C in a humidified atmosphere of 5% CO2 in air. For the experiments, cells were seeded at 35000/cm2 to obtain full confluence 24 h later. Differentiation to myotubes was induced by shifting confluent cultures to DMEM supplemented with 2% horse serum. The medium was changed every 2nd day, and within 5 days most of the cells were fused to form myotubes. Cells were then exposed to either Ad-CMV-GFP or Ad-cSTAT3-GFP (Vector Biolabs, Philadelphia, PA, USA) or treated with IL-6 (100ng/ml; R&D Systems, Minneapolis, MN, USA) for up to 48 h. Samples were then collected after each time point and used for further analyses.
Aliquots (150 µl) of culture supernatant from C2C12 myotubes exposed to IL-6 were collected at every time point. Fibrinogen levels were then assayed using the AssayMax Mouse Fibrinogen ELISA kit (AssayPro, St. Charles, MO, USA) following the instructions provided by the manufacturer.
All samples were stored at −80°C until tested (Rules Based Medicine, Austin, TX). Platelet poor plasma samples were thawed at room temperature, vortexed, spun at 13,000 x g for 5 minutes for clarification and 150 µL was removed into a master microtiter plate. Each sample was introduced into the capture microsphere multiplexes of the RodentMAP 2.0, thoroughly mixed and incubated at room temperature for 1 hour. Multiplexed biotinylated reporter antibodies were then added, mixed and incubated for an hour at room temperature. Multiplexes were developed using an excess of streptavidin-phycoerythrin solution. Analysis was performed in a Luminex 100 instrument and the resulting data stream was interpreted using proprietary data analysis software developed at Rules-Based Medicine and licensed to Qiagen Instruments. Unknown values for each of the analytes localized in a specific multiplex were determined using 4 and 5 parameter, weighted and non-weighted curve fitting algorithms included in the data analysis package.
Total RNA was extracted from flash frozen quadriceps using TRIzol as previously described
Biotinylated cRNA was prepared using the Illumina TotalPrep RNA Amplification Kit (Ambion, Inc., Austin, TX) according to the manufacturer's instructions, starting with 400 ng total quadriceps RNA. Successful cRNA generation was checked using the Bioanalyzer 2100. Samples were added to the BeadChip after randomization using the randomized block design to reduce batch effects. Hybridization to the MouseWG-6 v2.0 Expression BeadChips (Illumina, Inc., San Diego, CA), washing and scanning were performed according to the Illumina BeadStation 500 manual (revision C). The resulting raw microarray data were generated using Illumina BeadStudio. GeneSpring GX 7.3 was used for data normalization, statistical analysis (ANOVA, t-test) and hierarchical clustering. Only genes that were detected present (Illumina detection call p<0.01) in at least one group (control, moderate cachexia or severe cachexia) were included in the analysis. NextBio Professional and GeneGo Metacore were used for gene and pathway analysis. Genomatix Bibliosphere was used to generate a list of STAT3 associated genes.
All microarray data are MIAME compliant and have been deposited in the Gene Expression Omnibus (GEO) Database (NCBI) as Series GSE24112. Reviewers can access the data anonymously at the following link:
Total protein extract was obtained by homogenizing either skeletal muscle or C2C12 myotube samples in RIPA buffer (150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0) added with a protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Nuclear extracts resulted from homogenization of muscle tissue in ice cold 10 mM HEPES, pH 7.5, containing 10 mM MgCl2, 5mM KCl, 0.1 mM EDTA pH 8.0, 0.1% Triton X-100, 0.1 mM phenylmethanesulfonyl fluoride [PMSF], 1 mM DTT, 2 µg/ml aprotinin, 2 µg/ml leupeptin. Samples were then centrifuged (5 min, 3000 g), pellets resuspended in ice cold 20 mM HEPES, pH 7.9, containing 25% glycerol, 500 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, pH 8.0, 0.2 mM PMSF, 0.5 mM DTT, 2 µg/ml aprotinin, 2 µg/ml leupeptin, and incubated on ice for 30 min. Cell debris were removed by centrifugation (5 min, 3000 g) and the supernatant collected and stored at –80°C. Protein concentration (for both total and nuclear extracts) was determined using the Bradford protein assay method (Thermo Fisher Scientific, Suwanee, GA, USA).
Either total or nuclear protein extracts (30 µg) were then electrophoresed in gradient SDS gels. Gels were transferred to nitrocellulose membranes. Membranes were blocked with 1X TBS, 0.1% Tween-20 (TBST) with 5% w/v Bovine Serum Albumin (BSA) at room temperature for 1 hour, followed by an overnight incubation with diluted antibody in blocking buffer at 4°C with gentle shaking. After washing with TBST, the membrane was incubated at room temperature for 1 hour with a goat polyclonal anti-rabbit IgG secondary antibody conjugated to horseradish peroxidase (HRP) (sc-2313, Santa Cruz Biotechnology Inc., Santa Cruz, CA). Membranes were visualized with enhanced chemiluminescence (Pierce SuperSignal Pico or Femto) followed by exposure to film. Antibodies were pSTAT3 (9145), STAT3 (9132), GAPDH (2118), Histone H3 (4499) from Cell Signaling (Beverly, MA), and SOCS3 (Abcam 3693), Fibrinogen (Dako A0080), and SAA1 (R & D Systems AF2948). Mouse fibrinogen for quantitation was from Oxford Biomedical Research.
Cryosections (8 µm) from gastrocnemius muscles of both controls and C26-bearing mice were fixed in 3% formaldehyde, permeabilized in PBS-Triton 0.1% and incubated with pSTAT3 primary antibody (9145, Cell Signaling) overnight at 4°C. After three washes in PBS (5 min each), sections were incubated with the fluorescent secondary antibody (Alexa Fluor 488, Invitrogen, Carlsbad, CA, USA) in common antibody diluent (BioGenex, San Ramon, CA, USA) for 1 h at room temperature (RT) in the dark. After being washed three times with PBS (5 min each), sections were counterstained with DAPI and mounted with ProLong Gold Antifade mounting medium (Invitrogen, Carlsbad, CA, USA). Images were collected with constant exposure time across samples.
All results were expressed as means ± SEM. Representative Western blots show independent samples. Quantitation of the band intensities was performed using the ImageJ software (US National Institutes of Health, Bethesda, MD, USA). Significance of the differences was evaluated by analysis of variance (ANOVA) followed by Tukey's test for experiments with more than 2 groups or by Student t-test between 2 groups.
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The authors thank Donna O. McCarthy for cell lines, and Toumy Guettouche and the Oncogenomics Core Facility of the University of Miami for assistance with the microarray studies.