Journal: PLoS ONE

Article Title: Functional Substitution of a Eukaryotic Glycyl-tRNA Synthetase with an Evolutionarily Unrelated Bacterial Cognate Enzyme

doi: 10.1371/journal.pone.0094659

Figure Lengend Snippet: Oligomeric stryctures of glycyl-tRNA synthetase and the discriminator base N73 of tRNAs Gly .

Article Snippet: Aminoacylation reactions were carried out at 25°C in a buffer that contains 50 mM HEPES (pH 7.5), 50 mM KCl, 15 mM MgCl 2 , 5 mM dithiothreitol, 10 mM ATP, 0.1 mg/ml bovine serum albumin (BSA), 100 μM unfractionated yeast tRNA (Boehringer Mannheim, Germany) or 5 μM in vitro -transcribed yeast tRNA Gly , and 24 μM glycine (4 μM 3 H-glycine; Moravek Biochemicals, Brea, CA).

Techniques:

Journal: PLoS ONE

Article Title: Functional Substitution of a Eukaryotic Glycyl-tRNA Synthetase with an Evolutionarily Unrelated Bacterial Cognate Enzyme

doi: 10.1371/journal.pone.0094659

Figure Lengend Snippet: ( A ) Sequence homology among various GlyRSs. ( B ) Identity elements of tRNA Gly . Nucleotides and base pairs that were shown to be important for aminoacylation by GlyRS are boxed in E. coli and yeast cytoplasmic tRNAs Gly . The discriminator base N73 of tRNA Gly is marked with an arrow. Yeast cytoplasmic and mitochondrial tRNA Gly isoacceptors both possess the eukaryote-specific discriminator base A73, while E. coli tRNA Gly contains the bacterium-specific discriminator base U73.

Article Snippet: Aminoacylation reactions were carried out at 25°C in a buffer that contains 50 mM HEPES (pH 7.5), 50 mM KCl, 15 mM MgCl 2 , 5 mM dithiothreitol, 10 mM ATP, 0.1 mg/ml bovine serum albumin (BSA), 100 μM unfractionated yeast tRNA (Boehringer Mannheim, Germany) or 5 μM in vitro -transcribed yeast tRNA Gly , and 24 μM glycine (4 μM 3 H-glycine; Moravek Biochemicals, Brea, CA).

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Functional Substitution of a Eukaryotic Glycyl-tRNA Synthetase with an Evolutionarily Unrelated Bacterial Cognate Enzyme

doi: 10.1371/journal.pone.0094659

Figure Lengend Snippet: Aminoacylation activities of purified recombinant GlyRSs were determined by measuring the relative amounts of 3 H-glycine that were incorporated into tRNA using a liquid scintillation counter. ( A ) Glycylation of unfractionated E. coli tRNAs. ( B ) Glycylation of unfractionated yeast tRNAs. ( C ) Glycylation of in vitro -transcribed yeast tRNA n Gly . ( D ) Glycylation of in vitro -transcribed yeast tRNA m Gly . The final concentration of GlyRS used in the reactions was 5, 20, or 100 nM as indicated in the parenthesis. Data were obtained from three independent experiments and averaged.

Article Snippet: Aminoacylation reactions were carried out at 25°C in a buffer that contains 50 mM HEPES (pH 7.5), 50 mM KCl, 15 mM MgCl 2 , 5 mM dithiothreitol, 10 mM ATP, 0.1 mg/ml bovine serum albumin (BSA), 100 μM unfractionated yeast tRNA (Boehringer Mannheim, Germany) or 5 μM in vitro -transcribed yeast tRNA Gly , and 24 μM glycine (4 μM 3 H-glycine; Moravek Biochemicals, Brea, CA).

Techniques: Purification, Recombinant, In Vitro, Concentration Assay

Journal:

Article Title: Antiviral effect of the mammalian translation initiation factor 2? kinase GCN2 against RNA viruses

doi: 10.1038/sj.emboj.7601073

Figure Lengend Snippet: SV RNA stimulates GCN2-mediated phosphorylation of eIF2α. Purified wild-type GCN2 (GCN2-WT), the HisRS mutant (GCN2-m2) and PKR were assayed for their ability to phosphorylate eIF2α in the absence or the presence of increasing concentrations of uncharged bovine liver tRNA (Sigma) (A) or of poly(I)–poly(C) (Sigma) (B), as indicated. Phosphoproteins were analyzed by SDS–PAGE and autoradiography. Note an unknown phosphoprotein above the eIF2α band (A), which seems to be the consequence of the unspecific copurification of a kinase activity bound to TALON affinity resin. (C) In vitro eIF2α kinase assay of purified GCN2-WT, GCN2-K618R or GCN2-m2, in the absence or presence of SV RNA. Proteins were resolved into 10% SDS–PAGE and transferred to an immobilon-P membrane. The membrane was exposed to autoradiography (upper panels) and then probed with different antisera to detect eIF2α phosphorylated at serine 51 (eIF2α-P), total eIF2α and phosphorylated, and total GCN2 (lower panels) as indicated. (D) Kinase reactions were performed in the presence of the indicated concentrations of SV RNA or GCN2 RNA (as a negative control) and analyzed as described in (A). (E) Phosphorylation of eIF2α by Drosophila PERK, or mammalian PKR, GCN2 and HRI in the absence or presence of either poly(I)–poly(C) (1 μg/ml) or SV RNA (2.5 μg/ml, 0.64 nM). The analysis was carried out as described in (A).

Article Snippet: Purified wild-type GCN2 (GCN2-WT), the HisRS mutant (GCN2-m2) and PKR were assayed for their ability to phosphorylate eIF2α in the absence or the presence of increasing concentrations of uncharged bovine liver tRNA (Sigma) ( A ) or of poly(I)–poly(C) (Sigma) ( B ), as indicated.

Techniques: Purification, Mutagenesis, SDS Page, Autoradiography, Copurification, Activity Assay, In Vitro, Kinase Assay, Negative Control

Journal: Cell Death & Disease

Article Title: ID1-induced p16/IL6 axis activation contributes to the resistant of hepatocellular carcinoma cells to sorafenib

doi: 10.1038/s41419-018-0926-x

Figure Lengend Snippet: a The sensitivity of five HCC cell lines to sorafenib was measured by MTT assay. Cells were seeded in 96-well plates. After overnight incubation, cells were treated with different concentrations of sorafenib (5, 10, 20, 40 μM) for 24 h, then MTT assay was performed. DMSO was used as the control. The results were shown as inhibition rate, which indicates the percentage of cell growth inhibition caused by sorafenib treatment. b IC50 value was calculated based on the MTT results. The value represents the drug concentration of inducing 50% growth suppression compared to control cells. c HepG2 and Hep3B cells were incubated with sorafenib for 24 h, cell apoptosis was evaluated through flow cytometry (upper). The apoptosis ratio was calculated as the early apoptosis (lower right quadrant) plus the late apoptosis (upper right quadrant) percentage (lower). d HepG2 and Hep3B cells were incubated with β-gal staining solution. Senescent cells exhibited blue staining (left). Percentages of SA-β-gal positive cells were calculated and exhibited as a histogram (right). e Expression level of p16 in HepG2 and Hep3B cell lines was tested by western blot (left) and qRT-PCR (right). f IL6 expression in HepG2 and Hep3B cell lines was measured by qRT-PCR (left) and ELISA (right). For ELISA experiment, cells were seeded in 6-well plates and cultured for 24 h, then cell supernatants were collected. * p < 0.05; ** p < 0.01; *** p < 0.001, compared with relevant controls (Ctrl) or HepG2. The scale bars represent 25 μm. All immunoblots indicate molecular size markers in kDa

Article Snippet: ID1 overexpression plasmid pcDNA3 hId1(Cat No: #16061) and p16 overexpression plasmid pCMV p16 INK4A (Cat No: #10916) were purchased from Addgene.

Techniques: MTT Assay, Incubation, Control, Inhibition, Concentration Assay, Flow Cytometry, Staining, Expressing, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Cell Death & Disease

Article Title: ID1-induced p16/IL6 axis activation contributes to the resistant of hepatocellular carcinoma cells to sorafenib

doi: 10.1038/s41419-018-0926-x

Figure Lengend Snippet: a HepG2 and Hep3B cells were transfected with p16 overexpression vector and siRNA targeting p16, respectively. After 24 h, cells were incubated with different concentrations of sorafenib for 24 h. MTT assay was conducted to test cell viability. b After cultured with normal medium (Ctrl) or serum-free medium (Starvation) for 24 h, HepG2 cells were stained with β-gal solution. In Starvation group, senescent cells that exhibited blue staining were frequently observed under microscope. c p16 expression was detected by western blot. d IL6 concentration in cell supernatant was measured by ELISA. e MTT assay was used to determine the different effects of sorafenib on cell viability in HepG2 with routine culture (control group) or serum depletion (starvation group). f , g After cultured with normal medium (Ctrl) or serum-free medium (Starvation), cells were transfected with siRNA targeting p16 for 24 h ( f ) or pretreated with IL6 neutralizing antibody (5 ng/ml) for 2 h ( g ), and then incubated with 10 μM sorafenib for 24 h. MTT assay was conducted to test cell viability. NC: negative control. h In a transwell co-culture system, parent HepG2 cells were seeded in upper chamber, and the cells transfected with pCMV-p16 or empty vector were seeded in the bottom of wells. After 48 h-co-culture, the supernatant in bottom cells was collected to detect IL6 concentration. i The parent HepG2 cells in upper chamber were incubated with different concentrations of sorafenib for 24 h. Then cell viability was tested by MTT. * p < 0.05; ** p < 0.01; *** p < 0.001, compared with control (Ctrl). The scale bars represent 25 μm. All immunoblots indicate molecular size markers in kDa

Article Snippet: ID1 overexpression plasmid pcDNA3 hId1(Cat No: #16061) and p16 overexpression plasmid pCMV p16 INK4A (Cat No: #10916) were purchased from Addgene.

Techniques: Transfection, Over Expression, Plasmid Preparation, Incubation, MTT Assay, Cell Culture, Staining, Microscopy, Expressing, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Control, Serum Depletion, Negative Control, Co-Culture Assay

Journal: Cell Death & Disease

Article Title: ID1-induced p16/IL6 axis activation contributes to the resistant of hepatocellular carcinoma cells to sorafenib

doi: 10.1038/s41419-018-0926-x

Figure Lengend Snippet: a , b HepG2 cells transfected with siRNA against ID1 (Si-ID1) for 48 h. a RNA was isolated to detect ID1 and p16 to evaluate knockdown efficiency and its impact on p16 mRNA expression. b IL6 concentrations were quantified by ELISA (left). Percentages of SA-β-gal positive cells were calculated (right). c , d Hep3B cells transfected with pcDNA-ID1 for 24 h. c ID1 overexpression efficiency and its impact on p16 mRNA expression were determined by qRT-PCR. d IL6 concentrations were quantified by ELISA (left). Percentages of SA-β-gal positive cells were calculated (right). e A negative correlation between ID1 and p16 mRNA expression was observed in 24 HCC patient samples. f The levels of IL6, which were measured by ELISA, were higher in HCC patients with low concentrations of ID than those with high ones. * p < 0.05; ** p < 0.01; *** p < 0.001, compared with relevant controls (Ctrl) or empty pcDNA

Article Snippet: ID1 overexpression plasmid pcDNA3 hId1(Cat No: #16061) and p16 overexpression plasmid pCMV p16 INK4A (Cat No: #10916) were purchased from Addgene.

Techniques: Transfection, Isolation, Knockdown, Expressing, Enzyme-linked Immunosorbent Assay, Over Expression, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: ID1-induced p16/IL6 axis activation contributes to the resistant of hepatocellular carcinoma cells to sorafenib

doi: 10.1038/s41419-018-0926-x

Figure Lengend Snippet: a HepG2-SOR1 and HepG2-SOR2 were incubated with sorafenib for 24 h, MTT assay was employed to observe the efficacy. b The expression of ID1, p16, and IL6 mRNA in sorafenib-resistant cell lines was measured by qRT-PCR. c The number of positive SA-β-gal stained cells in sorafenib-resistant cell lines was determined. d Cells were seeded in 6-well plates and cultured for 24 h. Cell supernatant were collected and the secreted IL6 proteins were quantified by ELISA. e Changes of ID1, p16, and p-AKT(473) protein expression in HepG2-SOR1 was detected. f HepG2 cells were incubated with the conditioned medium from HepG2-SOR1 for 24 h. An obvious activation of p-AKT(473) was detected. g HepG2 cells incubated with the supernatant of HepG2 SOR1 were pre-treated with or without IL6 blocking antibody. The difference of sorafenib efficacy in the cells treated with the supernatant alone or the supernatant plus IL6 blocking was confirmed by MTT assay. h 24 h after transfection of pcDNA-ID1 plasmid or empty vector, HepG2 SOR1 cells were pretreated with LY294002 (5 μM) for 1 h prior to co-treatment with sorafenib (20 μM) for 24 h following by MTT assay. Inhibitory rate was analyzed through comparing the average absorbance value in treated cells to control cells. i 24 h after transfection of pcDNA-ID1 plasmid or empty vector, HepG2 SOR1 cells were pretreated with neutralizing antibody against IL6 (5 ng/ml) for 2 h prior to co-treatment with sorafenib (20 μM) for 24 h following by MTT assay. Inhibitory rate was analyzed through comparing the average absorbance value in treated cells to control cells. * p < 0.05; ** p < 0.01; *** p < 0.001. All immunoblots indicate molecular size markers in kDa

Article Snippet: ID1 overexpression plasmid pcDNA3 hId1(Cat No: #16061) and p16 overexpression plasmid pCMV p16 INK4A (Cat No: #10916) were purchased from Addgene.

Techniques: Incubation, MTT Assay, Expressing, Quantitative RT-PCR, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Activation Assay, Blocking Assay, Transfection, Plasmid Preparation, Control, Western Blot