Journal: Protein Science : A Publication of the Protein Society

Article Title: Molecular basis of EWS interdomain self‐association and its role in condensate formation

doi: 10.1002/pro.70316

Figure Lengend Snippet: EWS LCD forms interdomain contacts with RNA‐binding domain (RBD) motifs. (a) RNA‐binding protein EWS (EWS) domain architecture, net charge of each domain, and amino acid distribution. The EWS::FLI1 breakpoint (264) is marked with a black triangle; red and blue lines are negatively and positively charged residues. (b) Average pairwise interchain van der Waals contact map shown as −ln( n contact,ij / n max ), where n contact,ij is the average pairwise residue index interchain van der Waals contact and n max is the maximum values among all n contact,ij . (c) Summary of interdomain contact strength between EWS domain pairs represented as −ln(Σ n contact,ij /(Σ n contact,ij ) max ). (d) Fraction of total EWS interdomain contacts from co‐existence coarse grain (CG) simulations of EWS. Values represent the fraction of total interdomain contacts formed between specific domain combinations, calculated by summing all n contact,ij within each domain pair and normalizing by the total interdomain contacts across all domain combinations. FLI1, friend leukemia integration 1; LCD, low‐complexity domain; RGG, arginine–glycine–glycine; RRM, RNA‐recognition motif; ZnF, zinc finger, nuclear localization sequence.

Article Snippet: NMRPipe processing scripts are available upon reasonable request; expression plasmids were deposited at Addgene: EWS LCD (180467), EWS 1–120 (180464), EWS 99–199 (180465), EWS 171–264 (180466), EWS RGG1‐RRM (238369), EWS RRM‐RGG2 (188046), and EWS RGG3 (238370).

Techniques: RNA Binding Assay, Residue, Sequencing

Journal: Protein Science : A Publication of the Protein Society

Article Title: Molecular basis of EWS interdomain self‐association and its role in condensate formation

doi: 10.1002/pro.70316

Figure Lengend Snippet: RNA‐binding protein EWS (EWS) RNA‐binding domain (RBD) motifs impact condensate formation propensity of EWS LCD . (a) Turbidity measurements, (b) bright field microscopy, scale bar = 20 μm, and (c) condensate partitioning of 50 μM EWS LCD only (black) and + 50 μM of EWS RGG1‐RRM (orange), EWS RRM‐RGG2 (green), or EWS RGG3 (purple). Lighter shade is with 150 mM NaCl. (d) Phase diagram for EWS LCD + EWS RRM‐RGG2 (filled circles = visible condensates). Turbidity measurements of EWS LCD + EWS RRM‐RGG2 across different (e) pH, scale bar = 10 μm, and (f) NaCl concentrations. (g) Fluorescence microscopy of select [NaCl] from (f), scale bar = 20 μm. P ‐values: * P < 0.05, ** P < 0.005. Error bars = standard deviation. LCD, low‐complexity domain; RGG, arginine–glycine–glycine; RRM, RNA‐recognition motif.

Article Snippet: NMRPipe processing scripts are available upon reasonable request; expression plasmids were deposited at Addgene: EWS LCD (180467), EWS 1–120 (180464), EWS 99–199 (180465), EWS 171–264 (180466), EWS RGG1‐RRM (238369), EWS RRM‐RGG2 (188046), and EWS RGG3 (238370).

Techniques: RNA Binding Assay, Microscopy, Fluorescence, Standard Deviation

Journal: Protein Science : A Publication of the Protein Society

Article Title: Molecular basis of EWS interdomain self‐association and its role in condensate formation

doi: 10.1002/pro.70316

Figure Lengend Snippet: EWS LCD interacts with EWS RRM‐RGG2 through contacts distributed across their chains. Chemical shift perturbations (CSPs) (top left panels), average per‐residue contacts (bottom left panels), and normalized average pairwise van der Waals contacts (right panels) for (a) 15 N‐EWS 1–120 (cyan), (b) 15 N‐EWS 91–199 (pink), and (c) 15 N‐EWS 171–264 (green) interacting with EWS RRM‐RGG2 . CSPs are the maximum observed after addition of 3:1 molar ratio of EWS RRM‐RGG2 and color‐filled if CSPs >1 standard deviation (dashed black line). Asterisks indicate overlapped/ambiguous assignments (red) or prolines (black). Average per‐residue and pairwise van der Waals contacts were calculated from atomistic simulations of EWS RRM‐RGG2 with each EWS LCD fragment and are colored filled if contact values are > the mean for all three fragments (dashed black line). The average per‐residue contacts formed between EWS LCD fragments and EWS RRM‐RGG2 are shown along the left and bottom margins of each contact map. EWS, RNA‐binding protein EWS; RGG, arginine–glycine–glycine; RRM, RNA‐recognition motif.

Article Snippet: NMRPipe processing scripts are available upon reasonable request; expression plasmids were deposited at Addgene: EWS LCD (180467), EWS 1–120 (180464), EWS 99–199 (180465), EWS 171–264 (180466), EWS RGG1‐RRM (238369), EWS RRM‐RGG2 (188046), and EWS RGG3 (238370).

Techniques: Residue, Standard Deviation, RNA Binding Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Molecular basis of EWS interdomain self‐association and its role in condensate formation

doi: 10.1002/pro.70316

Figure Lengend Snippet: RNA‐recognition motif (RRM) and RGG2 form interdomain contacts with EWS LCD fragments. Chemical shift perturbations (CSPs) (top panels) and line broadening (bottom panels) of 15 N‐EWS RRM‐RGG2 titrated with (3:1 molar ratio) (a) EWS 1–120 , (b) EWS 91–199 , or (c) EWS 171–264 . CSPs (line broadening) > (<) 1 standard deviation (dashed line) are plotted in cyan (EWS 1–120 ), pink (EWS 91–199 ), or green (EWS 171–264 ). Asterisks mark unassigned/ambiguous/broadened residues (red) and proline (black). Unassigned glycine (G) or unclassified (R/X) resonances are plotted (right panels). EWS, RNA‐binding protein EWS; RGG, arginine–glycine–glycine.

Article Snippet: NMRPipe processing scripts are available upon reasonable request; expression plasmids were deposited at Addgene: EWS LCD (180467), EWS 1–120 (180464), EWS 99–199 (180465), EWS 171–264 (180466), EWS RGG1‐RRM (238369), EWS RRM‐RGG2 (188046), and EWS RGG3 (238370).

Techniques: Standard Deviation, RNA Binding Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Molecular basis of EWS interdomain self‐association and its role in condensate formation

doi: 10.1002/pro.70316

Figure Lengend Snippet: RNA‐recognition motif (RRM) and RGG2 form interdomain contacts with the low‐complexity domain (LCD) in full‐length RNA‐binding protein EWS (EWS). (a) Normalized average pairwise van der Waals contacts from single chain atomistic simulations of full‐length EWS. (b) Average per‐residue contacts between EWS LCD and EWS RRM‐RGG2 . Contacts formed by EWS LCD regions, comparing full‐length EWS versus individual LCD fragments in fragment simulations (top) and contacts formed by EWS RRM‐RGG2 , comparing interactions with LCD in full‐length EWS versus with individual LCD fragments in fragment simulations (bottom). EWS LCD,FL (extracted from atomistic simulations of single chain full‐length EWS, black), EWS 1–120 (cyan), EWS 91–199 (pink), or EWS 171–264 (green). (c) Average pairwise interdomain residue type contacts between EWS LCD and EWS RRM‐RGG2 extracted from single chain atomistic simulations of full‐length EWS; backbone–backbone (red), backbone–sidechain (blue), and sidechain–sidechain (green), first residue is from EWS LCD , second residue is from EWS RRM‐RGG2 . (d) Examples of EWS LCD ‐EWS RRM‐RGG2 ensemble images (EWS LCD , blue; RRM, yellow; RGG2, red) from atomistic simulations of full‐length EWS. RGG, arginine–glycine–glycine.

Article Snippet: NMRPipe processing scripts are available upon reasonable request; expression plasmids were deposited at Addgene: EWS LCD (180467), EWS 1–120 (180464), EWS 99–199 (180465), EWS 171–264 (180466), EWS RGG1‐RRM (238369), EWS RRM‐RGG2 (188046), and EWS RGG3 (238370).

Techniques: RNA Binding Assay, Residue

Journal: Molecular carcinogenesis

Article Title: CtBP2 Regulates Wnt Signal Through EGR1 to Influence the Proliferation and Apoptosis of DLBCL Cells.

doi: 10.1002/mc.23901

Figure Lengend Snippet: FIGURE 5 | Interfering with EGR1 inhibited the proliferation and cycle acceleration of DLBCL cells and promotes apoptosis. The EGR1 gene in cells was silenced through siRNA transfection. (A) The interference level of EGR1 was measured by western blot analysis. (B) Cell proliferation was detected by CCK8 assay. (C) EdU staining was performed to evaluate the level of cell proliferation; Flow cytometry was conducted to analyze the cell cycle (D) and the level of apoptosis (E). (F) The expressions of CtBP2 and apoptosis‐related proteins were detected by western blot. ***p < 0.001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: The antibodies utilized in this study included CtBP2 (10346‐1‐AP; Proteintech), EGR‐1 (22008‐1‐AP; Proteintech), Bcl‐2 (12789‐1‐AP; Proteintech), Bax (ab32503; Abcam), Caspase 3 (9961, CST), cleaved‐Caspase 3 (9962, CST), DKK1 (ab307367; Abcam), β‐catenin (ab32572; Abcam) and c‐Myc (ab185656; Abcam).

Techniques: Transfection, Western Blot, CCK-8 Assay, Staining, Flow Cytometry

Journal: Molecular carcinogenesis

Article Title: CtBP2 Regulates Wnt Signal Through EGR1 to Influence the Proliferation and Apoptosis of DLBCL Cells.

doi: 10.1002/mc.23901

Figure Lengend Snippet: FIGURE 6 | Interfere with CtBP2 inhibited the proliferation and cycle acceleration of DLBCL cells and promoted apoptosis through EGR1. The cells were transfected with an Ov‐EGR1 overexpression plasmid. (A) The overexpression level of EGR1 was assessed using Western blot analysis. (B) Cell proliferation was evaluated by CCK‐8 assay. (C) Cell proliferation was measured through EdU staining; Flow cytometry was performed to analyze the cell cycle (D) and the level of apoptosis (E). (F) The expression of apoptosis‐related proteins was detected by western blot assay. ***p < 0.001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: The antibodies utilized in this study included CtBP2 (10346‐1‐AP; Proteintech), EGR‐1 (22008‐1‐AP; Proteintech), Bcl‐2 (12789‐1‐AP; Proteintech), Bax (ab32503; Abcam), Caspase 3 (9961, CST), cleaved‐Caspase 3 (9962, CST), DKK1 (ab307367; Abcam), β‐catenin (ab32572; Abcam) and c‐Myc (ab185656; Abcam).

Techniques: Transfection, Over Expression, Plasmid Preparation, Western Blot, CCK-8 Assay, Staining, Flow Cytometry, Expressing

Journal: Molecular carcinogenesis

Article Title: CtBP2 Regulates Wnt Signal Through EGR1 to Influence the Proliferation and Apoptosis of DLBCL Cells.

doi: 10.1002/mc.23901

Figure Lengend Snippet: FIGURE 7 | EGR1 may be involved in the regulation of the Wnt/β‐catenin signaling pathway by CtBP2. The expressions of Wnt/β‐catenin signaling‐related proteins DKK1, β‐catenin and c‐MyC were detected by western blot. ***p < 0.001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: The antibodies utilized in this study included CtBP2 (10346‐1‐AP; Proteintech), EGR‐1 (22008‐1‐AP; Proteintech), Bcl‐2 (12789‐1‐AP; Proteintech), Bax (ab32503; Abcam), Caspase 3 (9961, CST), cleaved‐Caspase 3 (9962, CST), DKK1 (ab307367; Abcam), β‐catenin (ab32572; Abcam) and c‐Myc (ab185656; Abcam).

Techniques: Western Blot

Journal: Frontiers in Aging Neuroscience

Article Title: A Novel Egr-1-Agrin Pathway and Potential Implications for Regulation of Synaptic Physiology and Homeostasis at the Neuromuscular Junction

doi: 10.3389/fnagi.2017.00258

Figure Lengend Snippet: (A) In-silico analysis of human AGRN on chromosome 1 using the UCSC Genome Browser and Qiagen D-Code program identified several putative early growth response-1 (Egr-1) binding sites on the AGRN gene locus, both upstream and downstream of the TSS. (B) Primers were designed encompassing the putative binding sites, and four specific primer sets were used to amplify parts of the AGRN gene. (C) Chromatin immunoprecipitation (ChIP) assay was performed using these primer sets, and the Egr-1- AGRN interaction was determined using quantitative real time polymerase chain reaction (qPCR). Primers (P) 1, 3 and 4 showed binding activity, with the highest at region P3. Data was calculated as relative fold change, with the highest value set to 100. All results were normalized to internal levels of IgG and plotted relative to the binding activity of the negative control (NC) region from chromosome 1. n = 3. p -values were obtained using Student’s t -tests. *( p ≤ 0.05); **( p ≤ 0.01); ***( p ≤ 0.001); ****( p ≤ 0.0001).

Article Snippet: Human myc-tagged Egr-1 cDNA (OriGene) was cloned into PLVX-IRES-ZsGreen1 (Clontech Laboratories, Inc., Mountain View, CA, USA), a bi-cistronic lentiviral vector with a CMV promoter to create PLVX-IRES-ZsGreen1-Egr-1.

Techniques: In Silico, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Activity Assay, Negative Control

Journal: Frontiers in Aging Neuroscience

Article Title: A Novel Egr-1-Agrin Pathway and Potential Implications for Regulation of Synaptic Physiology and Homeostasis at the Neuromuscular Junction

doi: 10.3389/fnagi.2017.00258

Figure Lengend Snippet: Human M17 neuroblastoma cells were transiently transfected with pcDNA3.1 and pcDNA3.1Egr-1. (A) Representative western blot from three independent experiments. Egr-1 overexpression led to a significant decrease in agrin protein expression with respect to controls (empty vector, EV). β-actin was used as a loading control. (B) Rat primary hippocampal neurons were infected at 11 days in vitro (11 DIV) with lentiviral control (LV) or lentivirus containing myc-tagged human Egr-1 (LV-Egr-1) at an multiplicity of infection (MOI) of 20. Overexpression of Egr-1 led to a significant increase in EGR1 mRNA expression and decrease in AGRN mRNA expression with respect to LV controls. Values were normalized to GAPDH expression, and results were calculated using the ΔΔCT method and graphed as a fold-change of mRNA expression. n = 3. (C) Overexpression of myc-tagged LV-Egr-1 also led to a significant increase in myc and decrease in agrin protein expression. n = 3. (D) Next, rat primary hippocampal astrocytes were infected at 11 DIV with LV or LV-Egr-1 at an MOI of 20. Egr-1 overexpression did not significantly change agrin protein expression compared to LV controls in these cells. β-actin was used as a loading control. n = 3. (E) Mouse C2C12 myoblast cells were transiently transfected with pCMV6-XL5 (EV) and pCMV6-XL5-Egr-1(Egr-1). Overexpression of Egr-1 led to a significant increase in EGR1 and decrease in AGRN mRNA levels with respect to EV controls. n = 3. (F) When Egr-1 was overexpressed in C2C12 cells, Egr-1 protein levels were increased, and full length, 110 kDa, and 50 kDa agrin protein expression were all significantly decreased compared to EV controls. Coomassie staining was used as a loading control. n = 4. p -values were obtained using Student’s t -tests or 2-way analysis of variance (ANOVA), respectively. *( p ≤ 0.05); **( p ≤ 0.01); ***( p ≤ 0.001); ****( p ≤ 0.0001).

Article Snippet: Human myc-tagged Egr-1 cDNA (OriGene) was cloned into PLVX-IRES-ZsGreen1 (Clontech Laboratories, Inc., Mountain View, CA, USA), a bi-cistronic lentiviral vector with a CMV promoter to create PLVX-IRES-ZsGreen1-Egr-1.

Techniques: Transfection, Western Blot, Over Expression, Expressing, Plasmid Preparation, Infection, In Vitro, Staining

Journal: Frontiers in Aging Neuroscience

Article Title: A Novel Egr-1-Agrin Pathway and Potential Implications for Regulation of Synaptic Physiology and Homeostasis at the Neuromuscular Junction

doi: 10.3389/fnagi.2017.00258

Figure Lengend Snippet: RNA was extracted from the brains of seven wild type (WT) and seven Egr-1−/− mice, and mRNA was isolated. (A) qPCR showed that Egr-1−/− mouse brain has significantly more AGRN mRNA expression compared with WT mice. Protein samples were prepared from the frontal cortex, hippocampus and cerebellum of six WT, six Egr-1+/− and six Egr-1−/− mouse brains. The western blots presented are representative blots from three independent experiments. (B) Expression of the full length and 90 kDa agrin were significantly increased in the Egr-1−/− frontal cortex compared with that of WT. (C) In the hippocampus, full length, 110 kDa and 90 kDa agrin expression were significantly increased in Egr-1−/− mouse brain compared with WT. (D) In the cerebellum, full length and 90 kDa agrin expression were significantly increased in Egr-1−/− mouse brain compared with WT. p -values were obtained using Student’s t -tests or 2-way ANOVA, respectively. *( p ≤ 0.05); **( p ≤ 0.01); ***( p ≤ 0.001); ****( p ≤ 0.0001).

Article Snippet: Human myc-tagged Egr-1 cDNA (OriGene) was cloned into PLVX-IRES-ZsGreen1 (Clontech Laboratories, Inc., Mountain View, CA, USA), a bi-cistronic lentiviral vector with a CMV promoter to create PLVX-IRES-ZsGreen1-Egr-1.

Techniques: Isolation, Expressing, Western Blot

Journal: Frontiers in Aging Neuroscience

Article Title: A Novel Egr-1-Agrin Pathway and Potential Implications for Regulation of Synaptic Physiology and Homeostasis at the Neuromuscular Junction

doi: 10.3389/fnagi.2017.00258

Figure Lengend Snippet: RNA and protein samples were prepared from nine WT and nine Egr-1−/− mouse diaphragm and soleus muscles. The western blots presented are representative blots from three independent experiments. (A) AGRN mRNA expression was significantly increased in Egr-1−/− mouse diaphragm when compared with WT. Similarly, in both diaphragm (B,C) and soleus muscles (D,E) , the expression of agrin was significantly increased in Egr-1−/− mice compared with WT, with the exception of full length agrin in the soleus, which did not reach significance. All agrin protein bands were normalized to β-actin. Protein expression of neurotrypsin in the soleus was significantly decreased in Egr-1−/− mice compared with WT. All neurotrypsin protein bands were normalized to β-actin. p -values were obtained using Student’s t -tests or 2-way ANOVA, respectively. *( p ≤ 0.05); **( p ≤ 0.01); ***( p ≤ 0.001); ****( p ≤ 0.0001).

Article Snippet: Human myc-tagged Egr-1 cDNA (OriGene) was cloned into PLVX-IRES-ZsGreen1 (Clontech Laboratories, Inc., Mountain View, CA, USA), a bi-cistronic lentiviral vector with a CMV promoter to create PLVX-IRES-ZsGreen1-Egr-1.

Techniques: Western Blot, Expressing

Journal: Frontiers in Aging Neuroscience

Article Title: A Novel Egr-1-Agrin Pathway and Potential Implications for Regulation of Synaptic Physiology and Homeostasis at the Neuromuscular Junction

doi: 10.3389/fnagi.2017.00258

Figure Lengend Snippet: Diaphragms from five WT and five Egr-1−/− adult mice were whole mounted and stained with α-bungarotoxin to motor endplates made up of AchRs. (A) Representative 10× confocal images of WT and Egr-1−/− mouse diaphragm stained with α-bungarotoxin. (B) Representative 40× confocal images of WT and Egr-1−/− mouse diaphragm stained with α-bungarotoxin. The (C) density (total number) and (D) area of nerve innervation are significantly increased in Egr-1−/− compared with WT. The yellow arrows in the WT and Egr-1−/− panels highlight the width of the innervation band. (E) In Egr-1−/− mouse diaphragm, the area of individual endplates was significantly increased compared to WT. The size difference in the endplates is evident by comparing the identically-sized yellow boxes in the WT and Egr-1−/− panels. There was no significant difference in the thickness of the diaphragm, (F) or in the muscle fiber diameter, (G) in Egr-1−/− mouse diaphragm vs. WT. Z-stack images were quantified and analyzed to obtain results, and analysis was done using ImageJ software. p -values were obtained using Student’s t -tests. *( p ≤ 0.05); **( p ≤ 0.01); ***( p ≤ 0.001); ****( p ≤ 0.0001).

Article Snippet: Human myc-tagged Egr-1 cDNA (OriGene) was cloned into PLVX-IRES-ZsGreen1 (Clontech Laboratories, Inc., Mountain View, CA, USA), a bi-cistronic lentiviral vector with a CMV promoter to create PLVX-IRES-ZsGreen1-Egr-1.

Techniques: Staining, Software

Journal: Frontiers in Aging Neuroscience

Article Title: A Novel Egr-1-Agrin Pathway and Potential Implications for Regulation of Synaptic Physiology and Homeostasis at the Neuromuscular Junction

doi: 10.3389/fnagi.2017.00258

Figure Lengend Snippet: (A,B) Representative stacked confocal images of neuromuscular junctions (NMJs) from five WT and five Egr-1−/− mice soleus muscle NMJs. Soleus muscle preparations from WT and Egr-1−/− mice were labeled with anti-myosin heavy chain (anti-MHC) monoclonal antibodies to view Type I fibers (MHC-I; magenta), type IIa fibers (MHC-IIa; green), and then stained with α-bungarotoxin. n = 5. The mean number of surface endplates, (C) and NMJ fragments, (D) were increased in Egr-1−/− mice compared to WT. The % of smooth endplates (E) was decreased, and the % of faint NMJs was increased (F) in Egr-1−/− mouse soleus NMJs compared to WT. In WT vs. Egr-1−/− mice, there was no significant difference in the % of type I and type IIa soleus muscle fibers, (G) the weight of the soleus muscle, (H) or the overall bodyweight of the mice, (I) p -values were obtained using Student’s t -tests. *( p ≤ 0.05); **( p ≤ 0.01); ***( p ≤ 0.001); ****( p ≤ 0.0001).

Article Snippet: Human myc-tagged Egr-1 cDNA (OriGene) was cloned into PLVX-IRES-ZsGreen1 (Clontech Laboratories, Inc., Mountain View, CA, USA), a bi-cistronic lentiviral vector with a CMV promoter to create PLVX-IRES-ZsGreen1-Egr-1.

Techniques: Labeling, Staining

Journal: Frontiers in Aging Neuroscience

Article Title: A Novel Egr-1-Agrin Pathway and Potential Implications for Regulation of Synaptic Physiology and Homeostasis at the Neuromuscular Junction

doi: 10.3389/fnagi.2017.00258

Figure Lengend Snippet: Soleus muscle preparations from WT and Egr-1−/− mice were subjected to electrophysiological recordings. Twenty-one NMJs were recorded from five different WT mice, and 26 NMJs were recorded from five different Egr-1−/− mice. (A) Representation of spontaneous miniature endplate potential (MEPP) recordings from WT and Egr−/− mouse soleus NMJS. There was no significant difference in the mEPP frequency, (B) or amplitude, (C) in WT vs. Egr-1−/− mouse soleus NMJs. (D) Representation of Endplate potentials (EPPs) evoked by paired-pulse stimulation of the motor nerve (15 ms interval) from WT and Egr-1−/− mouse soleus NMJs. The EPP amplitude was similar in Egr-1−/− and WT mice, (E) but Egr-1−/− mice displayed an increase in quantal content (F,G) . There was no significant difference in the paired-pulse facilitation (PPF) between genotypes. To analyze the electrophysiology results according to fiber type, each NMJ recording was sorted according to fiber type and analyzed. There were no significant differences in the mEPP frequency, (H) or amplitude, (I) between genotypes in type I or type IIa fibers. There was no significance difference in EPP amplitude in either fiber type (J–L) , although the quantal content was increased in type IIa fibers (L,M) . There was no significant difference in PPF between genotypes when sorted by fiber type. p -values were obtained using Student’s t -tests or 2-way ANOVA, respectively. *( p ≤ 0.05); **( p ≤ 0.01); ***( p ≤ 0.001); ****( p ≤ 0.0001).

Article Snippet: Human myc-tagged Egr-1 cDNA (OriGene) was cloned into PLVX-IRES-ZsGreen1 (Clontech Laboratories, Inc., Mountain View, CA, USA), a bi-cistronic lentiviral vector with a CMV promoter to create PLVX-IRES-ZsGreen1-Egr-1.

Techniques:

Journal: Frontiers in Aging Neuroscience

Article Title: A Novel Egr-1-Agrin Pathway and Potential Implications for Regulation of Synaptic Physiology and Homeostasis at the Neuromuscular Junction

doi: 10.3389/fnagi.2017.00258

Figure Lengend Snippet: Four WT and four Egr-1−/− littermate adult mice were subjected to the open field test to measure general locomotion. The time spent moving, (A) was decreased in Egr-1−/− compared with WT mice and time spent at rest, (B) was increased. The Egr-1−/− moved a total distance that was less than WT mice, (C) and showed a decrease in the number discrete movement bouts, (D) the time spent in stereotypy movement, (E) and in discrete bouts of stereotypy, (F) when compared with WT mice. The same mice were subjected to the inverted grid test to measure strength and endurance in limb muscle. (G) Egr-1−/− 7458 show a significant decrease in limb strength and endurance compared to WT mice, as shown by the average latency in seconds that it took for the mice to fall from the inverted grid. p -values were obtained using Student’s t -tests. *( p ≤ 0.05); **( p ≤ 0.01); ***( p ≤ 0.001); ****( p ≤ 0.0001).

Article Snippet: Human myc-tagged Egr-1 cDNA (OriGene) was cloned into PLVX-IRES-ZsGreen1 (Clontech Laboratories, Inc., Mountain View, CA, USA), a bi-cistronic lentiviral vector with a CMV promoter to create PLVX-IRES-ZsGreen1-Egr-1.

Techniques:

Journal: Frontiers in Aging Neuroscience

Article Title: A Novel Egr-1-Agrin Pathway and Potential Implications for Regulation of Synaptic Physiology and Homeostasis at the Neuromuscular Junction

doi: 10.3389/fnagi.2017.00258

Figure Lengend Snippet: The Egr-1-agrin pathway may aid in the understanding of synaptic homeostatic mechanisms at the NMJ, which occur during the process of normal aging, as well as in the age-related condition known as sarcopenia. Reduction of Egr-1 expression leads to increased expression and cleavage of agrin in both brain and muscle as well as NMJ abnormalities, many of which are consistent with and reflective of aging and sarcopenia. Thus, dysregulation of the Egr-1-agrin pathway may provide a novel model system to study synaptic homeostasis at the NMJ, and Egr-1 deficient mice may serve as a suitable model to elucidate mechanisms associated with the underlying physiology of aging.

Article Snippet: Human myc-tagged Egr-1 cDNA (OriGene) was cloned into PLVX-IRES-ZsGreen1 (Clontech Laboratories, Inc., Mountain View, CA, USA), a bi-cistronic lentiviral vector with a CMV promoter to create PLVX-IRES-ZsGreen1-Egr-1.

Techniques: Expressing

Journal: bioRxiv

Article Title: A novel viral protein translation mechanism reveals mitochondria as a target for antiviral drug development

doi: 10.1101/2020.10.19.344713

Figure Lengend Snippet: (A) Genomic characteristics including histone modification and transcription factor binding status of tRNA TRL-TAA4-1 which matched with rare codon Leu-TTA was analysed using the UCSC database. (B) Overlap analysis of potential TRL-TAA regulatory transcription factors (TFs) and genes that increased in SARS-CoV-2 infection in A549-ACE2 and Calu3 cells using Venny2.1. (C) Heat map demonstrating the expression of 25 TFs in SARS-CoV-2-infected cell lines created using R studio. (D) Western Blot assay to assess the protein level of S after co-transfection with EGR1 or ATF2 in HEK-293 cells. (E). Differentially expressed genes after SARS-CoV-2 infection (GSE147507) were analyzed with R Studio. Pathway enrichment of highly expressed genes was mapped using Metascape (F) Distribution of genes including ACE2, TMPRSS2, ATF2 and identified TFs in human lung cells was created using public single cell sequence data.

Article Snippet: Human EGR1 clone with flag tag was bought from origene (RC209956), and the human ATF2 expressing clone had been constructed in our previous study( ).

Techniques: Modification, Binding Assay, Infection, Expressing, Western Blot, Cotransfection, Sequencing

Journal: bioRxiv

Article Title: A novel viral protein translation mechanism reveals mitochondria as a target for antiviral drug development

doi: 10.1101/2020.10.19.344713

Figure Lengend Snippet: (A) The interaction network of proteins that bound with S was analyzed using Metascape. (B) The frequency of rare codons in SARS-CoV-2, S protein, human nuclear genome and the human mitochondrial genome using the Codon Usage Database. (C) The protein expression of S flanked by SARS-CoV-2 5’- and 3’-UTR sequences in HEK-293 cell lines. (D) The effect of EGR1 and ATF2 on S protein (plus SARS-CoV-2 5’- and 3’ UTS sequences) expression was analysed using Western blot after transfection into HEK-293 cells. Fold change was determined using Image J software. (E) The skeleton of recombinant plasmids encoding S with or without mitochondrial localisation signals (MLS) RnaseP or RMRP. (F) Protein expression of SARS-CoV-2 derived sequences including S, E and ORF8 and variants expressing MLS as analysed by Western blot after transfection into HEK-293 cells. (G) The effect of EGR1 and ATF2 on wild type spike with RMRP binding motif (MRP).

Article Snippet: Human EGR1 clone with flag tag was bought from origene (RC209956), and the human ATF2 expressing clone had been constructed in our previous study( ).

Techniques: Expressing, Western Blot, Transfection, Software, Recombinant, Derivative Assay, Binding Assay