Lls in some conditions [39,40]. Although our results indicated involvement of mNanog in Activin/nodal signaling, they also suggested that mNanog contributes, at least in part, to the gene regulation mechanism around Activin/nodal signaling that underpins mesoderm formation in Xenopus. We expect that other factors involved with pluripotency, like Oct3/4 and Sox2, could also induce activity similar to that observed with mNanog, although our preliminary findings showed no mesoderm gene induction following coinjection with xSox2 or Oct61 (data not shown). This study sought to identify the Xenopus gene homolog of mammalian Nanog by using sequences of axolotl and newt [41,42]. Although we designed six primers in homeodomain and caspase domain (Fig. S1 and M M section) and performed seven rounds of degenerate PCR using combination of these primers, we failedDorsal Mesoderm-Inducing Activity of NanogFigure 4. Dorsal mesoderm induction by mNanog was involved with inhibition of BMP signaling. A) Target genes of BMP signaling were inhibited by mNanog injection, based on the expressions of Xvent1 (1st column), Xvent2 (2nd column), BMP4 (3rd column), and ODC (4th column). 0 pg (lane 3, 4), 200 pg (lane 5), or 400 pg (lane 6) of mNanog was ��-Sitosterol ��-D-glucoside biological activity injected into animal poles, which were treated with 10 ng/ml of Activin A (lane 4?6) at stage 9. ACs were harvested at stage 11. B) Co-injection analysis with Xvent2 mRNA. 200 pg of mNanog (lane 2?) and 0 pg (lane 3), 500 pg (lane 4), 1 ng (lane 5), or 2 ng (lane 6) of Xvent2 were co-injected into animal poles at the 2-cell stage. ACs were dissected at stage 9 and homogenized at stage 11 for RNA preparation. The expressions of several dorsal mesoderm genes (chd, gsc, xlim-1) and BMP4 were analyzed. C) Effect of cycloheximide (CHX) on the induction of mesoderm genes by mNanog. 0 pg (lane 1, 2) or 400 pg (lane 3, 4) of mNanog was injected into animal poles at the 2-cell stage, 0 mg/ml (lane 1, 3) or 40 mg/ml (lane 3, 4) of CHX was added. D) Model of expected mechanism of mesoderm gene induction by mNanog. “X” indicates presumptive factor(s) for regulating both Xvent1/2 and Xnr1/2 expression by mNanog. doi:10.1371/journal.pone.0046630.gto find any sequence identified as xNanog, although many identified were similar genes including Xvent1 (6/16) and Hoxd11 (6/16) (Fig. S1). Moreover, whole genome analysis of Xenopus tropicalis revealed no known nucleotide sequence for the XtNanog gene. Further exploration of Xenopus Nanog or another factor that substitutes for Nanog is obviously needed.Table S1 The summary of phenotypes in embryos injected with mNanog into AP region. (DOCX)AcknowledgmentsWe thank to Dr. Shuji Takahashi, Dr. Yoshikazu Haramoto, and Prof. Tsutomu Kinoshita for critical discussion. We also thank Dr. Moritoshi Sato for technical supports. Mouse cDNA for mNanog cloning was a kind gift of Dr. Yuko Aihara.Supporting InformationFigure S1 Summary of the degenerative PCR for cloning of 12926553 the Xenopus Nanog gene. Upper panel: schematic diagram of Nanog protein. CD, HD, and WR indicate the caspase domain, homeodomain, and tryptophan-rich domain, respectively. U1–2 and L1? indicate primer positions for the PCR. Lower panel: summary of degenerative PCR results. In Ex.6, we performed PCR with an amplified Linolenic acid methyl ester site product using the U2 and L1 primers as a template. The number of obtained gene fragments is also shown. (TIF)Author ContributionsConceived and designed the experiments: TM. Performed the experiments: TM AM KI SY SN.Lls in some conditions [39,40]. Although our results indicated involvement of mNanog in Activin/nodal signaling, they also suggested that mNanog contributes, at least in part, to the gene regulation mechanism around Activin/nodal signaling that underpins mesoderm formation in Xenopus. We expect that other factors involved with pluripotency, like Oct3/4 and Sox2, could also induce activity similar to that observed with mNanog, although our preliminary findings showed no mesoderm gene induction following coinjection with xSox2 or Oct61 (data not shown). This study sought to identify the Xenopus gene homolog of mammalian Nanog by using sequences of axolotl and newt [41,42]. Although we designed six primers in homeodomain and caspase domain (Fig. S1 and M M section) and performed seven rounds of degenerate PCR using combination of these primers, we failedDorsal Mesoderm-Inducing Activity of NanogFigure 4. Dorsal mesoderm induction by mNanog was involved with inhibition of BMP signaling. A) Target genes of BMP signaling were inhibited by mNanog injection, based on the expressions of Xvent1 (1st column), Xvent2 (2nd column), BMP4 (3rd column), and ODC (4th column). 0 pg (lane 3, 4), 200 pg (lane 5), or 400 pg (lane 6) of mNanog was injected into animal poles, which were treated with 10 ng/ml of Activin A (lane 4?6) at stage 9. ACs were harvested at stage 11. B) Co-injection analysis with Xvent2 mRNA. 200 pg of mNanog (lane 2?) and 0 pg (lane 3), 500 pg (lane 4), 1 ng (lane 5), or 2 ng (lane 6) of Xvent2 were co-injected into animal poles at the 2-cell stage. ACs were dissected at stage 9 and homogenized at stage 11 for RNA preparation. The expressions of several dorsal mesoderm genes (chd, gsc, xlim-1) and BMP4 were analyzed. C) Effect of cycloheximide (CHX) on the induction of mesoderm genes by mNanog. 0 pg (lane 1, 2) or 400 pg (lane 3, 4) of mNanog was injected into animal poles at the 2-cell stage, 0 mg/ml (lane 1, 3) or 40 mg/ml (lane 3, 4) of CHX was added. D) Model of expected mechanism of mesoderm gene induction by mNanog. “X” indicates presumptive factor(s) for regulating both Xvent1/2 and Xnr1/2 expression by mNanog. doi:10.1371/journal.pone.0046630.gto find any sequence identified as xNanog, although many identified were similar genes including Xvent1 (6/16) and Hoxd11 (6/16) (Fig. S1). Moreover, whole genome analysis of Xenopus tropicalis revealed no known nucleotide sequence for the XtNanog gene. Further exploration of Xenopus Nanog or another factor that substitutes for Nanog is obviously needed.Table S1 The summary of phenotypes in embryos injected with mNanog into AP region. (DOCX)AcknowledgmentsWe thank to Dr. Shuji Takahashi, Dr. Yoshikazu Haramoto, and Prof. Tsutomu Kinoshita for critical discussion. We also thank Dr. Moritoshi Sato for technical supports. Mouse cDNA for mNanog cloning was a kind gift of Dr. Yuko Aihara.Supporting InformationFigure S1 Summary of the degenerative PCR for cloning of 12926553 the Xenopus Nanog gene. Upper panel: schematic diagram of Nanog protein. CD, HD, and WR indicate the caspase domain, homeodomain, and tryptophan-rich domain, respectively. U1–2 and L1? indicate primer positions for the PCR. Lower panel: summary of degenerative PCR results. In Ex.6, we performed PCR with an amplified product using the U2 and L1 primers as a template. The number of obtained gene fragments is also shown. (TIF)Author ContributionsConceived and designed the experiments: TM. Performed the experiments: TM AM KI SY SN.
Related Posts
Experiments have been executed in triplicate, and the error bars are SE values for 3 samples.ten% FBS have been utilized in all experiments
Foreseeable future research of the in vivo GC response of ASM may possibly assist explain the variations among in vitro and in vivo study final results. Our review has determined well-acknowledged and much more novel GC responsive genes, but it was also subject matter to restrictions. We used cell lines from 4 white male topics, […]
Levels of MRP1 were reported to increase after exposure to oxidative stress inducing agents
ed. So far, the reliable list of inhibitory mediators is limited to IL-10, TGF-, NO and PGE2. Our results are in agreement with previous findings: inhibitory activity of supernatants obtained from DCreg educated on lung stroma partly depended upon the presence of PGE2, and the differences in regulatory activity of DCreg obtained from nave and […]
His apparent lack of conflict. Indeed, other authors reported that emotionalHis apparent lack of conflict.
His apparent lack of conflict. Indeed, other authors reported that emotionalHis apparent lack of conflict. Certainly, other authors reported that emotional conflicts specifically come for the fore when experts notify patients about their treatment allocation [25]. Furthermore, EasterPLOS 1 DOI:0.37journal.pone.055940 May perhaps 9,0 Patients’ and Professionals’ Representation of Placebo in RCTsand coworkers (2006) reported that, […]