Journal: BMC Research Notes

Article Title: On the cytotoxicity of HCR-NTPase in the neuroblastoma cell line SH-SY5Y

doi: 10.1186/1756-0500-2-102

Figure Lengend Snippet: Positions of siRNAs used to silence HCR-NTPase in SH-SY5Y . ClustalW-alignment of the eight most diverse sequences out of a group of 29 species that have been found to contain Walker A and B motifs similar to those of HCR-NTPase . The sequences were from Homo sapiens , Mus musculus , Drosophila melanogaster , Aquifex aeolicus (aaTHEP1), Thermophilum pendens , Methanopyrus kandleri , an uncultured archaeon, Sulfolobus tokodaii , and Aeropyrum pernix , respectively.

Article Snippet: HCR-NTPase as a fusion with red fluorescent protein [ ] (HCR-NTPase-RFP) was overexpressed using the plasmid pmaxFP-Red-C (Amaxa, Cologne, Germany).

Techniques:

Journal: BMC Research Notes

Article Title: On the cytotoxicity of HCR-NTPase in the neuroblastoma cell line SH-SY5Y

doi: 10.1186/1756-0500-2-102

Figure Lengend Snippet: Silencing HCR-NTPase in SH-SY5Y has no effect . Normalised cell counts (in % of untransfected control) of SH-SY5Y transfected with plasmids containing GFP, RFP, siRNA1, siRNA2, siRNA3, and siRNA4, respectively. The data for the GFP- and RFP-controls are the same as those shown in table 1. The error bars indicate the standard deviations of at least 3 independently performed experiments.

Article Snippet: HCR-NTPase as a fusion with red fluorescent protein [ ] (HCR-NTPase-RFP) was overexpressed using the plasmid pmaxFP-Red-C (Amaxa, Cologne, Germany).

Techniques: Transfection

Journal: BMC Research Notes

Article Title: On the cytotoxicity of HCR-NTPase in the neuroblastoma cell line SH-SY5Y

doi: 10.1186/1756-0500-2-102

Figure Lengend Snippet: Cytotoxicity of HCR-NTPase expressed in SH-SY5Y . Phase contrast and fluorescence images of SH-SY5Y cells are shown. The cells were either untreated (A) or transfected with Plasmids encoding HCR-NTPase (B), GFP (C), GFP + HCR-NTPase (D), RFP (E), and an HCR-NTPase-RFP fusion product (F).

Article Snippet: HCR-NTPase as a fusion with red fluorescent protein [ ] (HCR-NTPase-RFP) was overexpressed using the plasmid pmaxFP-Red-C (Amaxa, Cologne, Germany).

Techniques: Fluorescence, Transfection

Journal: BMC Research Notes

Article Title: On the cytotoxicity of HCR-NTPase in the neuroblastoma cell line SH-SY5Y

doi: 10.1186/1756-0500-2-102

Figure Lengend Snippet: Quantitative analysis of the cytotoxicity of HCR-NTPase expressed in SH-SY5Y

Article Snippet: HCR-NTPase as a fusion with red fluorescent protein [ ] (HCR-NTPase-RFP) was overexpressed using the plasmid pmaxFP-Red-C (Amaxa, Cologne, Germany).

Techniques:

Journal: BMC Research Notes

Article Title: On the cytotoxicity of HCR-NTPase in the neuroblastoma cell line SH-SY5Y

doi: 10.1186/1756-0500-2-102

Figure Lengend Snippet: Rescue of SH-SY5Y cells by RNA-interference . Normalised cell counts (in % of untransfected control) of SH-SY5Y transfected with plasmids encoding a fusion protein between HCR-NTPase and RFP alone (HCR-NTPase-RFP) or cotransfected with siRNA1, siRNA2, siRNA3, and siRNA4, respectively. Compared to the control, less than 1% of cells containing the HCR-NTPase-RFP fusion protein could be detected by fluorescence microscopy (data not shown). The data for the GFP-, RFP- and HCR-NTPase-RFP-controls are the same as those shown in table 1. The error bars indicate the standard deviations of at least 3 independently performed experiments.

Article Snippet: HCR-NTPase as a fusion with red fluorescent protein [ ] (HCR-NTPase-RFP) was overexpressed using the plasmid pmaxFP-Red-C (Amaxa, Cologne, Germany).

Techniques: Transfection, Fluorescence, Microscopy

Journal: BMC Research Notes

Article Title: On the cytotoxicity of HCR-NTPase in the neuroblastoma cell line SH-SY5Y

doi: 10.1186/1756-0500-2-102

Figure Lengend Snippet: Cytotoxicity of the E114A-HCR-NTPase mutant and rescue of SH-SY5Y cells by siRNA3 . Normalised cell counts (in % of untransfected control) of SH-SY5Y transfected with plasmids encoding the E114A-HCR-NTPase mutant (E114A) alone or cotransfected with either GFP or siRNA3. Whereas fGFP + E114A were analysed by fluorescence microscopy, all other cells were counted via phase contrast microscopy. The data for the GFP-, RFP- and HCR-NTPase-controls are the same as those shown in table 1. The error bars indicate the standard deviations of at least 3 independently performed experiments.

Article Snippet: HCR-NTPase as a fusion with red fluorescent protein [ ] (HCR-NTPase-RFP) was overexpressed using the plasmid pmaxFP-Red-C (Amaxa, Cologne, Germany).

Techniques: Mutagenesis, Transfection, Fluorescence, Microscopy

Journal: BMC Research Notes

Article Title: On the cytotoxicity of HCR-NTPase in the neuroblastoma cell line SH-SY5Y

doi: 10.1186/1756-0500-2-102

Figure Lengend Snippet: Effect of Z-VAD-FMK on the cytotoxicity induced by HCR-NTPase and E114A-HCR-NTPase in SH-SY5Y

Article Snippet: HCR-NTPase as a fusion with red fluorescent protein [ ] (HCR-NTPase-RFP) was overexpressed using the plasmid pmaxFP-Red-C (Amaxa, Cologne, Germany).

Techniques:

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Enhanced Salt Tolerance of Rhizobia-inoculated Soybean Correlates with Decreased Phosphorylation of the Transcription Factor GmMYB183 and Altered Flavonoid Biosynthesis *

doi: 10.1074/mcp.RA119.001704

Figure Lengend Snippet: Phosphorylation motifs enriched from differentially phosphorylated peptides after salt treatment. A, and B, are phosphorylation motifs extracted from the phosphopeptides in the Up group and Down group, respectively, when the soybean roots were inoculated with rhizobia. C, and D, show phosphorylation motifs extracted from the phosphopeptides in the Up and Down groups when the rhizobia-inoculated root was treated with NaCl. E, GmMYB183 could be a substrate of GmCK2α. Phosphorylation assay was carried out as described in the experimental procedures. WT and S61D: recombinant proteins of the N-terminal 168 aa of GmMYB183-S61D and GmMYB183-WT fused to a Trx- and a 6 x His-tags at its N terminus, and a 6 x His-tag at its C terminus. Data indicated that the Ser61 is the unique site in GmMYB183.

Article Snippet: The subcellular localization of the GmMYB183-RFP fusion protein was observed using a Zeiss LSM710 confocal microscope ( C ).

Techniques: Phosphorylation Assay, Recombinant

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Enhanced Salt Tolerance of Rhizobia-inoculated Soybean Correlates with Decreased Phosphorylation of the Transcription Factor GmMYB183 and Altered Flavonoid Biosynthesis *

doi: 10.1074/mcp.RA119.001704

Figure Lengend Snippet: GmMYB183-RFP localizes in the nucleus of soybean root cells. The original roots of soybean seedling were removed and infected with A. rhizogenes carrying a GmMYB183-RFP expressing construct, transgenic roots with red fluorescence were selected using a Nikon SMZ1500 fluorescence stereoscope (A, B). The subcellular localization of the GmMYB183-RFP fusion protein was observed using a Zeiss LSM710 confocal microscope (C).

Article Snippet: The subcellular localization of the GmMYB183-RFP fusion protein was observed using a Zeiss LSM710 confocal microscope ( C ).

Techniques: Infection, Expressing, Construct, Transgenic Assay, Fluorescence, Microscopy

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Enhanced Salt Tolerance of Rhizobia-inoculated Soybean Correlates with Decreased Phosphorylation of the Transcription Factor GmMYB183 and Altered Flavonoid Biosynthesis *

doi: 10.1074/mcp.RA119.001704

Figure Lengend Snippet: GmMYB183 binds to a MYB-specific Cis-element present in the promoter of GmCYP81E11. A, Distribution of MYB binding motifs (G/A/T)(G/A/T)T(C/A)(A/G)(A/G)(G/T)(T/A) in the promoter of GmCYP81E11. Position of the probe (underlined sequence) used for ChIP-based binding assay is shown below the gene. Position Weight Matrix of MYB binding motifs was curated in the JASPAR database (http://jaspar.genereg.net/cgi-bin/jaspar_db.pl?rm=browse&db=core&tax_group=plants). B, and C, ChIP-qPCR assays indicates that GmMYB183 binds to the promoter sections containing the MYB-binding motifs in vivo. The empty vector was used as a negative control (NC). An asterisk indicates a significant difference (p ≤ 0.05) to the NC according to Student's t test. FC means fold change. D, and E, EMSA assays showed that GmMYB183 specially binds to the GmCYP81E11-P1 fragment from the GmCYP81E11 promoter (D), and phosphorylation at S61 of GmMYB183 enhances this interaction (E). GmCYP81E11-P1 (ttttATGTATTAGTGATTAAGTTTAATAACGTGA) or a mutated version with its ATTAAGTT core sequence changed to ATAAAGTT (GmCYP81E11-P1M) was labeled as a probe, and 200 or 500 folds of unlabeled double strand GmCYP81E115-P1 fragment was set as the competitor. F, and G, The transcription levels of GmMYB183 (F) and GmCYP81E11 (G) in soybean transgenic roots expressing empty vector (EV), GmMYB183-overexpression (GmMYB183-OE) and RNAi (GmMYB183-KD) constructs, respectively. Data presented are mean ± S.E. (n = 3). H, Transcription of GmMYB183 in soybean roots treated with R(-)Na(-), R(+)Na(-), R(-)Na(+) and R(+)Na(+). Data represent mean values ± S.E., each sample was analyzed with three biological replicates. An asterisk indicates a significant difference (p ≤ 0.05, Student's t test) between treatment [R(+)Na(-), R(-)Na(+) and R(+)Na(+)] and the control [R(-)Na(-)].

Article Snippet: The subcellular localization of the GmMYB183-RFP fusion protein was observed using a Zeiss LSM710 confocal microscope ( C ).

Techniques: Binding Assay, Sequencing, In Vivo, Plasmid Preparation, Negative Control, Labeling, Transgenic Assay, Expressing, Over Expression, Construct

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Enhanced Salt Tolerance of Rhizobia-inoculated Soybean Correlates with Decreased Phosphorylation of the Transcription Factor GmMYB183 and Altered Flavonoid Biosynthesis *

doi: 10.1074/mcp.RA119.001704

Figure Lengend Snippet: Ononin contents in OE or K D roots in response to salt stress

Article Snippet: The subcellular localization of the GmMYB183-RFP fusion protein was observed using a Zeiss LSM710 confocal microscope ( C ).

Techniques:

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Enhanced Salt Tolerance of Rhizobia-inoculated Soybean Correlates with Decreased Phosphorylation of the Transcription Factor GmMYB183 and Altered Flavonoid Biosynthesis *

doi: 10.1074/mcp.RA119.001704

Figure Lengend Snippet: HPLC-based analysis of ononin contents in transgenic roots of soybean. A, The chromatographic spectrum observed from ononin standard separation. B, to (N) The chromatographic spectrum observed from HPLC separation of transgenic GmCYP81E11-OE R(-)Na(-) (B), GmCYP81E11-OE R(+)Na(-) (C), GmCYP81E11-OE R(+)Na(+) (D), GmCYP81E11-KD R(-)Na(-) (E), GmCYP81E11-KD R(+)Na(-) (F), GmCYP81E11-KD R(+)Na(+) (G), GmMYB183 OE-WT R(-)Na(-) (H), GmMYB183 OE-S61D R(-)Na(-) (I), GmMYB183 OE-S61D R(+)Na(-) (J), GmMYB183 OE-S61A R(-)Na(-) (K), GmMYB183-KD R(-)Na(-) (L), GmMYB183-KD R(+)Na(-) (M), GmMYB183-KD R(+)Na(+) (N) roots.

Article Snippet: The subcellular localization of the GmMYB183-RFP fusion protein was observed using a Zeiss LSM710 confocal microscope ( C ).

Techniques: Transgenic Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Enhanced Salt Tolerance of Rhizobia-inoculated Soybean Correlates with Decreased Phosphorylation of the Transcription Factor GmMYB183 and Altered Flavonoid Biosynthesis *

doi: 10.1074/mcp.RA119.001704

Figure Lengend Snippet: ROS elimination capacity of OE or K D roots

Article Snippet: The subcellular localization of the GmMYB183-RFP fusion protein was observed using a Zeiss LSM710 confocal microscope ( C ).

Techniques:

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Enhanced Salt Tolerance of Rhizobia-inoculated Soybean Correlates with Decreased Phosphorylation of the Transcription Factor GmMYB183 and Altered Flavonoid Biosynthesis *

doi: 10.1074/mcp.RA119.001704

Figure Lengend Snippet: Effects of salt treatment on transgenic roots of composite soybean plants. Composite soybean plants were generated by infection with A. rhizogenes strain K599 carrying empty vector (EV), overexpression constructs of GmMYB183 (OE-WT), GmMYB183S61D (OE-S61D), GmMYB183S61A (OE-S61A) and RNAi-mediated knockdown construct of GmMYB183 (KD). Transgenic roots were confirmed by DsRed fluorescence as described in the materials and methods section. One-week old composite plants were treated in 1/4 fold Fahräeus medium without (control) or with 200 mM NaCl [NaCl (+)] for 4 weeks. A, Plants are typical representatives of three repeats of salt treatments. B, Fresh weights of transgenic roots: data expressed are means ± s.d. of root samples from three plants and the experiments are repeated three times with similar results; an asterisk indicates a significant difference from the control (EV) based on p ≤ 0.05 of student's t test;double asterisks indicates a significant difference from the control (EV) based on p ≤ 0.01 of student's t test.

Article Snippet: The subcellular localization of the GmMYB183-RFP fusion protein was observed using a Zeiss LSM710 confocal microscope ( C ).

Techniques: Transgenic Assay, Generated, Infection, Plasmid Preparation, Over Expression, Construct, Fluorescence

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Enhanced Salt Tolerance of Rhizobia-inoculated Soybean Correlates with Decreased Phosphorylation of the Transcription Factor GmMYB183 and Altered Flavonoid Biosynthesis *

doi: 10.1074/mcp.RA119.001704

Figure Lengend Snippet: The phosphorylated peptide GYAsxDDA in GmMYB183 protein is highly conserved in several plants. The peptide GYAsxDDA was found in homologous proteins of GmMYB183 in several plants including Arachis ipaensis, Medicago truncatula, Glycine max, Cajanus cajan, Phaseolus vulgaris, Vigna angularis, Prunus persica, Malus domestica, Morus notabilis and Citrus clementine.

Article Snippet: The subcellular localization of the GmMYB183-RFP fusion protein was observed using a Zeiss LSM710 confocal microscope ( C ).

Techniques:

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Enhanced Salt Tolerance of Rhizobia-inoculated Soybean Correlates with Decreased Phosphorylation of the Transcription Factor GmMYB183 and Altered Flavonoid Biosynthesis *

doi: 10.1074/mcp.RA119.001704

Figure Lengend Snippet: A hypothetical model for transcription factors GmMYB183 in regulating soybean's responses to salinity. After the perception of salinity or rhizobia inoculation signal, the phosphorylation of GmMYB183 was inhibited. Furthermore, the expression of its downstream gene GmCYP81E11 was downregulated and the GmCYP81E11-induced monohydroxy B-ring flavonoid (ononin) declined. Finally, the adjusted flavonoids appropriately reduced the ROS for enhancing the soybean's tolerance to salinity.

Article Snippet: The subcellular localization of the GmMYB183-RFP fusion protein was observed using a Zeiss LSM710 confocal microscope ( C ).

Techniques: Expressing