Photographs of the sample were taken at 1 h, 2 h, and 4 h. Other samples were studied in the same way. Based on the drug encapsulation efficiency, the same quantity of etoposide was applied to all formulations for the sedimentation

study. Determination of loading amount and in vitro release test The amount of incorporated etoposide was measured through UV–vis spectroscopy. A known weight of ECCNS sample was placed in a 10-mL flask, QNZ mw then 100 μL of 3 M HCl solution was subsequently added into it, and the flask was filled with 100% phosphate buffer solution (PBS) (pH = 7.4) until total volume reached 10 mL. After the ECCNS sample was totally dissolved, the concentration of etoposide was determined with a UV–vis spectrophotometer at 285 nm. The concentration of etoposide was calculated according to an already obtained calibrating curve Epoxomicin in vivo of etoposide (Abs = 0.00645c + 0.01599, r = 0.99923). The drug loading capacity and encapsulation efficiency are calculated as follows: The etoposide release test was performed in 180 mL PBS at pH 7.4,

5.8, and 3.0. ECCNS (25 mg) was resuspended in 10 mL PBS and loaded in a dialysis bag. The release system was swayed in a bath reciprocal shaker at 100 rpm and at constant temperature of 37°C above for 120 h. Aliquots (2 mL) were extracted at desired time intervals, and another 2 mL fresh PBS was added to the system. The accumulated amount of etoposide released was determined Silibinin by UV absorption at 285 nm. Cytotoxicity assay Cytotoxicity was characterized by MTT test through the human embryonic kidney (HEK) 293 T cells. 293 T cells with a density of 1 × 104 cells/well were seeded on a 96-well polystyrene plate, and each well contained 100 μL of DMEM (high glucose) medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution. Cells were subsequently

incubated at 37°C in a 5% CO2 humid incubator for 24 h. CCNSs, etoposide, and ECCNSs were added to the wells with concentrations 5, 10, 20, and 40 μg/mL in sequence. The HEK 293 T cells were incubated as described above for 24 and 48 h. A control experiment was performed with pure culture medium without treatment. Then, 20 μL (5 mg/mL) of MTT was added to each well, and the plate was further incubated for 4 h to deoxidize MTT under light-blocking condition. After removal of the MTT dye solution, cells were treated with 150 μL DMSO, and the absorbance at 490 nm was measured using ELX 800 UV reader (BioTek, Winooski, VT, USA), and cell viability was calculated by: Inhibition against SGC-7901 cells The antitumor ARN-509 research buy effect of CCNSs, etoposide, and ECCNSs against human gastric carcinoma (SGC-7901) cells was examined by cell viability test. SGC-7901 cells with a density of 8 × 104 cells/well were seeded on a 96-well polystyrene plate, and each well contained 100 μL of RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution.

Acknowledgements This work was supported by the Wellcome

Trust. L.B. Meakin and G.L. Galea are recipients of Integrated Training Fellowships for Veterinarians from the Wellcome Trust. Conflicts of interest None. Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, selleck distribution, and reproduction in any medium, provided the original author(s) and the source are credited. References 1. Suva LJ, Gaddy D, Perrien DS, Thomas RL, Findlay DM (2005) Regulation of bone mass by mechanical loading: microarchitecture and genetics. Curr Osteoporos Rep 3:46–51PubMedCrossRef 2. Skerry TM (2008) The response of bone to mechanical loading and disuse: fundamental principles and influences on osteoblast/osteocyte homeostasis. Arch Biochem Biophys 473:117–123PubMedCrossRef 3. Ozcivici E, Luu YK, AZD2014 Adler B, Qin YX, Rubin J, Judex S, Rubin CT (2010) Mechanical signals

as anabolic agents in bone. Nat Rev Rheumatol 6:50–59PubMedCrossRef 4. Bonewald LF, Johnson ML (2008) Osteocytes, mechanosensing and Wnt signaling. Bone 42:606–615PubMedCrossRef 5. Price JS, Sugiyama T, Galea GL, Meakin LB, Sunters A, Lanyon LE (2011) Role of endocrine and paracrine factors in the adaptation of bone to mechanical loading. Curr Osteoporos Rep 9:76–82PubMedCrossRef 6. Galea GL, Sunters A, Meakin LB, Pyruvate dehydrogenase Zaman G, Sugiyama T, Lanyon LE, Price JS (2011) Sost down-regulation by mechanical VS-4718 mw strain in human osteoblastic cells involves PGE2 signaling via EP4. FEBS Lett 585:2450–2454PubMedCrossRef 7. Pead MJ, Lanyon LE (1989) Indomethacin modulation of load-related stimulation of new bone formation in vivo. Calcif Tissue Int 45:34–40PubMedCrossRef 8. Chow JW, Chambers TJ (1994) Indomethacin has distinct early and late actions on bone formation induced by mechanical stimulation. Am J Physiol 267:E287–E292PubMed 9. Forwood MR (1996) Inducible cyclo-oxygenase (COX-2) mediates the induction of bone

formation by mechanical loading in vivo. J Bone Miner Res 11:1688–1693PubMedCrossRef 10. Li J, Burr DB, Turner CH (2002) Suppression of prostaglandin synthesis with NS-398 has different effects on endocortical and periosteal bone formation induced by mechanical loading. Calcif Tissue Int 70:320–329PubMedCrossRef 11. Alam I, Warden SJ, Robling AG, Turner CH (2005) Mechanotransduction in bone does not require a functional cyclooxygenase-2 (COX-2) gene. J Bone Miner Res 20:438–446PubMedCrossRef 12. Kohrt WM, Barry DW, Van Pelt RE, Jankowski CM, Wolfe P, Schwartz RS (2010) Timing of ibuprofen use and bone mineral density adaptations to exercise training. J Bone Miner Res 25:1415–1422PubMedCrossRef 13.

Gold-coated, reflective probes (NSG10) were used with an intermediate spring constant k = 11.5 N/m, a maximum tip radius of curvature of 10 nm, and a resonance frequency of 190 to 325 kHz (Europe MicroMasch, Tallinn, Estonia). Images were captured using the tapping mode at ambient conditions (room temperature 24°C ± 1°C and relative

humidity 38% ± 5%). After landing with tip on the sample surface, a damping ratio (A sp/A 0) of 0.5 to 0.6 and a line frequency of 0.25 to 0.6 Hz were optimized for imaging. The AFM was placed on a vibration isolation table (TS-150, Table Stable, Zwillikon, GDC-0941 supplier Switzerland) to eliminate external vibrational noise. Image processing and root-mean-square (RMS) roughness S q calculations were carried out using the scanning probe image processor program (SPIP™, Image Metrology A/S, Hørsholm, Denmark). Before calculation, images were plane-corrected and the ISO 11562 Gaussian profile filter was implemented. selleck products LXH254 results and discussion TiO2 nanoparticle coatings on paperboard exhibit superhydrophobicity (water contact angle above 160°) that can be converted into a highly hydrophilic surface (water contact angle below 20°) by ultraviolet (UV) illumination via the photocatalytic activity of TiO2

as presented in Figure 2. The crystalline form of the LFS-deposited TiO2 nanoparticles is mainly anatase [22], analyzed from the TEM diffraction pattern. UV light induces free radicals and photocatalytic oxidation that change the surface chemistry of nanoparticles from hydrophobic to Methamphetamine hydrophilic. In our previous study [13], we used X-ray photoelectron spectroscopy (XPS) to study the mechanisms of such wettability conversion: after the UV irradiation, increased values of both O/C and O/Ti ratios were observed. This corresponds to the increased amount of hydroxyl groups on the outermost TiO2 nanoparticle surface. Furthermore, our time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis

[14] was in agreement with the XPS results with decreased relative amounts of hydrocarbons after the UV irradiation. The surface superhydrophobicity can be recovered by a heat treatment. After the heat treatment, the O/C and O/Ti ratios decreased, and the highly resolved spectra of O 1s verified the decreased amount of oxygen related to the hydroxyl groups [13]. A similar change is observed in the ToF-SIMS spectra [14] with increased relative amounts of hydrocarbon chains originating from the volatile organic compounds used in the base paper substrate. We have previously shown that surface wettability can be alternated between wetting and non-wetting states for several cycles, and the observed changes in wettability correlate well with the changes in the surface chemistry of the TiO2 nanoparticle-coated surface [13, 14]. Figure 2 Water contact angles as a function of the number of calendering nips. For TiO2 nanoparticle-coated and the reference paperboard.

In addition to TPP, the negative groups on the surface

of ASNase II were counteracted with the positively charged -NH3 + groups of CS during the cross-linking process. Moreover, TPP could counteract with the positively charged -NH3 + groups on the surface of ASNase II and compact the enzyme both inside and on the surface of the particle. Particles possessing a zeta potential of about 20 to 25 mV may sometimes be considered relatively stable [37]. However, having a sufficient MDV3100 concentration zeta potential is extremely important for the role of nanoparticles as carriers for drugs or proteins; the nanoparticles must be capable of ionically holding active molecules or biomolecules. Nanoparticle used for the final characterization were loaded with 4 mg lyophilized ASNase II. Fourier transform infrared spectrometry analysis The FTIR spectra for ASNase II (a), CS (b), CSNPs (c), and ASNase see more II-loaded CSNPs (d) are shown in Figure 2. The peaks at CHIR98014 3,291 cm−1 in the ASNase II spectrum (a) and at 3,288 cm−1 in the CS spectrum (b) relate to the stretching of O-H and N-H bonds. In the CSNPs spectrum (c), a shift from 3,288 to 3,299 cm−1 is seen and the peak at 3,299 cm−1 becomes more intense; this indicates the -NH3 + interactions with TPP. A corresponding peak in the ASNase II-loaded CSNPs (d) at 3,294 cm−1 becomes wider; this effect is attributable to the participation

of ASNase II in hydrogen bonding and -NH group interactions [38]. In CSNPs, a new sharp peak appears at 1,409 cm−1 and the 1,594 cm−1 peak of -NH2 bending vibration shifts to 1,536 cm−1.

We suppose that the Gemcitabine phosphoric groups of TPP are linked with -NH3 + group of CS; inter- and intra-molecular interactions are enhanced in CSNPs [39]. A shift from 1,027 cm−1 to the sharper peak at 1,032 cm−1 corresponds to the stretching vibration of the P = O groups in CSNPs. Two peaks at 1,636 cm−1 (amide I bending) and 1,544 cm−1 (amide II bending) in ASNase II-loaded CSNPs correspond to the high intensity peaks at 1,638 and 1,536 cm−1 in the ASNase II spectra; this result proves successful loading of ASNase II in CSNPs and also indicates some interactions between CS with TPP and ASNase II [40]. Figure 2 FTIR spectra of (A) ASNase II, (B) CS, (C) CSNPs, and (d) ASNase II-loaded CSNPs. Morphology studies for the nanoparticles Figure 3 shows the TEM images of CSNPs and ASNase II-loaded CSNPs. From the TEM images, both CSNPs (Figure 3A) and ASNase II-loaded CSNPs (Figure 3B) are spherical and exist as discrete spheres, along with a few partial cohesive spheres. The dark core of nanoparticles is due to the fact that the staining reagent has penetrated through the particle. In Figure 3A, a fairly uniform size (the average size 250 ± 11 nm, PDI ~ 0.48) distribution and the smooth border around the CSNPs could be observed. In Figure 3B, ASNase II-loaded CSNPs exhibit an irregular surface with a core surrounded by a fluffy coat made of ASNase II.

The intensity of bands was quantitated by densitometry and is represented as the bar graph for cleaved PARP-1 (open bar) and cleaved this website caspase-3 (closed bar) after normalizing against α-tubulin expression. Data are representative of two independent experiments with similar results. Effect of gemcitabine, sorafenib and EMAP on animal survival In vivo animal survival studies in SCID-NOD mice resulted in a median survival (m.s.) of 22 days in the control group without treatment. Median animal survival was increased significantly after Gem (29 days, p=0.009 vs. control) but not after sorafenib (23 days, p=0.67 vs. control) or EMAP (25 days, p=0.11) monotherapy (Figure 5). Further improvement

in animal survival was encountered in the combination therapy groups Gem+So (m.s. 30 days, p=0.004 vs. controls), Gem+EMAP (m.s. 33 days, p=0.002 vs. controls) and Gem+So+EMAP (m.s. 36 days, p=0.004 vs. controls). Compared to the Gem monotherapy group, median survival was significantly higher in the Gem+EMAP (p=0.046) and Gem+So+EMAP therapy group (p=0.03) but not in the Gem+So therapy group (p=0.3). Survival in the So+EMAP therapy group (m.s. 24 days, p=0.18 vs. control) was not significantly different from controls or single agent therapy

learn more groups (Figure 5). No sign of BIBW2992 nmr drug-related toxicity was observed in any of the treatment groups. Figure 5 Effects of gemcitabine (Gem), sorafenib (So) and EMAP (E)

treatment on the overall survival of mice. AsPC-1 cells (0.75 x 106) were injected intraperitoneally in SCID mice and treatment started after 2 weeks with gemcitabine (100 mg/Kg, 2 times a week), sorafenib (30 mg/Kg, 5 times a week), and EMAP (80 μg/Kg, 5 times a week) for 2 weeks. The curve represents the survival time from the beginning of therapy. Discussion PDAC shows limited susceptibility to almost all classes of cytotoxic drugs. Several molecular genetic abnormalities in PDAC are being encountered with a high frequency, including activating K-ras mutation, loss of p16, p53 and DPC4 (deleted Thymidine kinase in pancreatic cancer, locus 4) function, and over-expression of multiple receptor tyrosine kinases [36, 37]. Tumor heterogeneity resulting from the diverse molecular abnormalities acquired during malignant transformation creates a rationale to evaluate multi-targeted therapeutic strategies against many human malignancies including PDAC. Sorafenib is a novel, potent, small molecular mass inhibitor with combined anticancer activities through the inhibition of tumor cell proliferation and tumor angiogenesis. Combining conventional cytotoxic drugs, such as gemcitabine, with targeted agents that specifically interfere with key operational pathways responsible for PDAC progression, such as sorafenib, is gaining more traction in the efforts to identify more effective combination treatments for PDAC.

A paper in this supplement [19] describes a recent development effort for GO terms, both general and specific, that describe processes involved https://www.selleckchem.com/products/pd-0332991-palbociclib-isethionate.html in the interactions between eukaryotic pathogens and their hosts. In the GO, the more general terms usually describe processes that are shared across diverse organisms, while more specific terms are often

created to describe organism-specific processes. For example one of the child terms of “”GO:0044406 adhesion to host”" is “”GO:0052001 type IV pili-dependent localized adherence to host”", a term relevant to bacterial symbionts. More recently added sibling terms to GO:0052001 include ones describing processes associated with adhesion of filamentous organisms to their host: “”GO:0075001 adhesion of symbiont infection structure to host”" and “”GO:0075004 adhesion of symbiont spore to host”" ([19] this supplement). Since the focus of PAMGO was primarily on microbial pathogens, initial term sets were generated to annotate genes in the microbe that are involved in interactions with the host, e.g. “”GO:0044405 recognition of host”". However,

it quickly became obvious that reciprocal terms that describe the interactions from LDC000067 the perspective of the host would also be required to meet all annotation needs (e.g. “”GO:0051855 recognition of symbiont”" Therefore, parallel sets of terms have been constructed to describe processes in the microbe as well as processes in the host that are involved in the interactions. In addition, terms were included to describe symbiotic relationships where neither organism could be clearly identified as “”host”" versus “”symbiont.”" Thus, under the GO term “”GO:0044419 interspecies interaction between organisms”", there are child

terms to accommodate symbiont genes that affect the host under “”GO:0051701 interaction with host”" and parallel terms appropriate for host genes under “”GO:0051702 interaction with symbiont”" (Figure 1). To learn more about these terms, including their definitions, CBL0137 chemical structure synonyms, child terms, and genes annotated using them, see [20] and search using the term or a keyword within the term. Annotation of selected microbial genomes with new and existing GO terms The members of the PAMGO consortium have been working on annotating the genomes of the bacteria Pseudomonas Sulfite dehydrogenase syringae pv tomato DC3000, Dickeya dadantii (Erwinia chrysanthemii) 3937, and Agrobacteriun tumefaciens; the fungus Magnaporthe oryzae (M. grisea); oomycete species. There are currently over 29,000 GO annotations as a result of the PAMGO project. The annotations can be viewed at [21]. As an example, Meng et al., [22] in this supplement report a comprehensive GO annotation of the rice pathogen Magnaporthe oryzae. In this paper, annotations were based on information from published literature as well as sequence-based analyses.

Thus, while the blood pH values are slightly EPZ015938 ic50 elevated for both Control and Experimental groups, the significant change in blood pH demonstrated by the Experimental group is likely a real effect of consuming AK water. Influence on Hydration Status Consumption of AK water following a

dehydrating bout of cycling exercise has previously been shown to rehydrate cyclists faster and more completely than the consumption of placebo bottled water (i.e., Aquafina) [8]. Following the consumption of AK water, the cyclists demonstrated less total urine output, their urine was more concentrated (higher specific gravity), and total blood protein concentration was lower, all of which are expected observations for improved hydration status [8]. Even though the present study was performed under free-living conditions, the Experimental group demonstrated an increased urine concentration (osmolality; Table 7), a decreased total urine output selleck inhibitor (Figure 1), as well as a decreased blood osmolality (Figure 2) by the end of the treatment period. These changes suggest that while SRWC was relatively stabile across measurement periods (Table 4), a relatively greater proportion of the AK water consumed during the treatment phase

was being retained within find more the cardiovascular system. Indeed, the cyclist hydration study described above [8] reported that water retention at the end of a 3-hour recovery period was 79.2 ± 3.9% when subjects drank AK water versus 62.5 ± 5.4% when drinking the placebo (P < 0.05). Thus, the present study has shown that the habitual consumption of mineralized Meloxicam bottled water can actually improve indicators of hydration status over non-mineralized bottled water under free-living conditions that is consistent with lab-controlled study results. Similar to what was described for changes in acid-base balance above, however, the onset of these observations did not begin with

the immediate consumption of AK water. In fact, changes in total urine output, urine osmolality, and blood osmolality did not appear to begin changing until the end of the first week of consuming AK water, with significant changes always occurring at the end of the second week of consumption. Unfortunately, the present study was designed to observe possible changes in acid-base balance and hydration status rather than decipher mechanistic causes. However, it is possible to speculate on some contributing causes given that the AK water manufacturer lists only three major naturally occurring minerals on the bottle label (Calcium at 2.8 mg/L, Silica at 16.0 mg/L, and Potassium at 23.0 mg/L) as well as the proprietary blend of mineral-based alkalizing supplement called Alka-PlexLiquid™. According to the manufacturer, Alka-PlexLiquid™ is a freely dissolvable form of a patented blend of mineral-based alkalizing ingredients called Alka-Plex™ granules.

Arch Biochem Biophys 1982, 213:395–404.PubMedCrossRef 14. Billington SJ, Jost BH, Cuevas WA, Bright KR, Songer JG: The Arcanobacterium ( Actinomyces ) pyogenes hemolysin, pyolysin, is a novel member of the thiol-activated cytolysin family. J Bacteriol 1997, 179:6100–6106.PubMed 15. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K: Current protocols in molecular biology. Volume 1. New York, NY: Greene Publishing Associates and John Wiley and Sons, Inc.; 1994. 16. Yasawong M, Teshima H, Lapidus A, Nolan M, Lucas S, Glavina Del Rio T, Tice H, Cheng JF, Bruce D, Detter

C, et al.: Complete genome sequence of Arcanobacterium haemolyticum type strain (11018). Stand Genomic Sci 2010,3(2):126–135.PubMedCrossRef 17. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller Thiazovivin supplier W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database

Belinostat in vitro search programs. Nucleic Acids Res 1997, 25:3389–3402.PubMedCrossRef 18. Lowe TM, Eddy SR: tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucl Acids Res 1997, 25:955–964.PubMedCrossRef 19. Nielsen H, Engelbrecht J, Brunak S, von Heijne G: Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 1997, 10:1–6.PubMedCrossRef 20. Zucker M: Mfold web buy CHIR98014 server for nucleic acid folding and hybridization prediction. Nucl Acids Res 2003, 31:3406–3415.CrossRef 21. Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994, 22:4673–4680.PubMedCrossRef 22. Rampersaud R, Planet PJ, Randis TM, Kulkarni R, Aguilar JL, Lehrer RI, Ratner AJ: Inerolysin, a cholesterol-dependent

cytolysin produced by Lactobacillus iners . Journal of Bacteriology 2011,193(5):1034–1041.PubMedCrossRef 23. Gelber SE, Aguilar JL, Lewis KL, Ratner AJ: Functional and phylogenetic characterization of Vaginolysin, MYO10 the human-specific cytolysin from Gardnerella vaginalis . Journal of Bacteriology 2008,190(11):3896–3903.PubMedCrossRef 24. Fernandez-Miyakawa ME, Jost BH, Billington SJ, Uzal FA: Lethal effects of Clostridium perfringens epsilon toxin are potentiated by alpha and perfringolysin-O toxins in a mouse model. Vet Microbiol 2007, 127:379–385.PubMedCrossRef 25. Jost BH, Trinh HT, Songer JG, Billington SJ: Immunization with genetic toxoids of the Arcanobacterium pyogenes cholesterol-dependent cytolysin, pyolysin, protects mice against infection. Infect Immun 2003, 71:2966–2969.PubMedCrossRef 26. Meyer F, Paarmann D, D’Souza M, Olson RD, Glass EM, Kubal M, Paczian T, Rodriguez A, Stevens R, Wilke A, et al.: The metagenomics RAST server – a public resource for the automatic phylogenetic and functional analysis of metagenomes. BMC Bioinformatics 2008, 9:386.PubMedCrossRef 27.

Total RNA from excised C57BL/6 mice skin was used as control. B16-F10 cells expressed mRNA of Sall4, Dppa5, Ecat1, c-Myc, Grb2, β-catenin, and Stat3, which were not expressed in control C57/BL6 skin samples. (B, C) Buparlisib ic50 B16-F1 (B) or B16-F10 cells (C) were injected subcutaneously into C57BL/6 mice. Seven days after the injection, the tumor was excised. Total RNA was extracted and RT-PCR was performed. Two additional experiments resulted in similar profiles to that shown here. Expression of ES-specific click here genes

during tumorigenesis Next, we examined the expression of ES-specific genes in B16 sublines during tumorigenesis. B16-F1 or B16-F10 cells were injected subcutaneously into C57BL/6 mice. Seven days after injection the tumor was excised and total RNA was extracted. RT-PCR analysis revealed that Ecat1, Dppa5, Ecat8, EPZ-6438 cell line GDF3, Sall4, Klf4, c-Myc, β-catenin, Stat3, and Grb2 were expressed after tumorigenesis of B16-F1 and/or B16-F10 (Figure 1B,C). Sall4, Grb2, β-catenin, and Stat3 are known to be expressed in tumor cells and their roles in cancer has been already studied [19, 27, 28]. Ecat1, Dppa5, and GDF3 genes are expressed in ES cells, but their expression in tumor has not yet been reported. We initially focused on Ecat1 and Dppa5 during tumorigenesis.

To investigate the expression kinetics we excised the B16-F1 or B16-F10 tumor 7, 10, or 14 days after implantation, and extracted total RNA. RT-PCR analysis revealed that Ecat1 and Dppa5 expression did not increase during tumorigenesis in both sublines (Figure 2A and 2B). Figure 2 Expression kinetics of Ecat1, Dppa5, and GDF3 during tumorigenesis. Histamine H2 receptor B16-F1 and B16-F10 cells were injected subcutaneously into C57BL/6 mice. Tumors were excised on the indicated day. Total RNA was extracted from the tumor and RT-PCR (A-D) or RT-qPCR (E, F) was performed to detect

Ecat1, Dppa5, and GDF3. (A, B) RT-PCR analyses revealed that mRNA of Eca1 and Dppa5 decreased during tumorigenesis. (C, E) In B16-F1 cells, GDF3 peaked at day 7 after tumor injection and then gradually decreased. (D, F). In contrast, GDF3 expression in B16-F10 cells increased 7 days after tumor injection and maintained a high level until 14 days after injection. Next, we focused on GDF3. GDF3 mRNA expression was not detectable in B16-F1 cells cultured in dish (day 0 in Figure 2C) and only a weak expression was detected in B16-F10 cells cultured in dish (day 0 in Figure 2D). Interestingly, GDF3 mRNA expression increased approximately 10-fold 7 days after s.c. inoculation in both B16-F1 and B16-F10 cells (Figure 2C and 2D). Following the increase for 7 days after injection, GDF3 expression gradually decreased in B16F1 cells, but maintained a high level in B16-F10 cells (Figure 2E and 2F). GDF3 promotes the tumorigenesis of B16 melanoma GDF3 is a member of TGF-β super family which is expressed in ES cells and in several human tumor cells. However, the role of GDF3 during tumorigenesis remains undetermined.

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