Journal: PLoS Pathogens

Article Title: Host signaling and EGR1 transcriptional control of human cytomegalovirus replication and latency

doi: 10.1371/journal.ppat.1008037

Figure Lengend Snippet: (A) Fibroblasts were infected with TB40/E GFP (MOI = 1) and put into serum-free media at 24hpi. Cells were stimulated with 10nM EGF at 48 hpi and cells were harvested between 1 and 24 hours post stimulation. Lysates were separated by SDS-PAGE, transferred and blotted with α- IE1/2, α- UL135 , α- UL138 , and α Tubulin. Protein levels from 4 replicates were normalized to no EGF treated control and 1h post EGF treatment is graphed. Statistical significance was calculated by student t-test; asterisks *** p-value < 0.001. Error bars represent SEM.(B) Graphical representation of putative EGR1 binding sites located within UL135 ORF starting at nt-306 and nt-896, in reference to UL135 start codon. P-values were calculated using PhysBinder prediction software. (C) A model depicting how EGFR signaling promotes EGR1 expression by either directing its expression through MEK/ERK signaling or by blocking FOXO1 suppression of EGR1 transcription though PI3K/AKT signaling. (D) CD34 + HPCs were infected with WT virus (MOI of 2) and treated with MEK/ERK (Binimetinib and SCH772984) or PI3K/AKT (LY294002 and MK-2206) inhibitors at 4 hpi. RNA was isolated at 48 hpi EGR1 measured by RT-qPCR relative to H6PD. Graph represents the average from two replicates with error bars that represent the range.

Article Snippet: EGR1 (44D5) , Rabbit , Cell Signaling , Western 1:1,000; ChIP 10 μL/ 4x10 6 cells.

Techniques: Infection, SDS Page, Binding Assay, Software, Expressing, Blocking Assay, Isolation, Quantitative RT-PCR

Journal: PLoS Pathogens

Article Title: Host signaling and EGR1 transcriptional control of human cytomegalovirus replication and latency

doi: 10.1371/journal.ppat.1008037

Figure Lengend Snippet: (A) Fibroblasts were transduced with EGR1 3xFlag lentivirus and then infected with WT or UL133/8 null mutant (negative control; NC) TB40/E virus (MOI = 1). Chromatin was immunoprecipitated (ChIP) with IgG control or antibodies specific to EGR1 or histone 3 (H3) and the presence of Site 1 or Site 2 was detected in the precipitates by PCR. As a positive control, PCR was also performed on 2% of the ChIP input. Gel is a representative experiment from 3 replicates. Diagram represents the amplicon region used for Site 1 and Site 2 detection. (B) ChIP-qPCR using SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling) was performed on fibroblasts infected for 48 h and pulsed with EGF for 1h, fibroblasts expressing EGR1 3xFlag infected for 48 h, or pure population of infect CD34 + HPCs in long-term culture for 5 days (6 dpi total). Fibroblasts were infected at an MOI of 1 and the CD34 + HPCs were infected at an MOI of 2. The presence of EGR1 Site 1 or Site 2 sequence was quantified by qPCR relative to a 2% input control and normalized to WT levels.

Article Snippet: EGR1 (44D5) , Rabbit , Cell Signaling , Western 1:1,000; ChIP 10 μL/ 4x10 6 cells.

Techniques: Transduction, Infection, Mutagenesis, Negative Control, Immunoprecipitation, Positive Control, Amplification, Chromatin Immunoprecipitation, Expressing, Sequencing

Journal: PLoS Pathogens

Article Title: Host signaling and EGR1 transcriptional control of human cytomegalovirus replication and latency

doi: 10.1371/journal.ppat.1008037

Figure Lengend Snippet: (A) Fibroblasts were transduced with either EGR1 3xFlag or empty vector control and infected with WT TB40/E GFP (MOI = 1) 24 hours later. At 48 hpi, protein lysates were separated by SDS-PAGE and proteins detected using α-FLAG, α- UL138 , and α-Tubulin. (B) HEK293T cells were co-transfected with EGR1 3xFlag , UL133/8 encoding plasmid, or empty vector controls (minus sign). Protein lysates were harvested at 48h, samples were separated by SDS-PAGE and proteins detected using α-FLAG, α- UL138 , and α-Tubulin. (A-B) UL138 protein levels from either 4(A) or 3(B) independent experiments were normalized to the control and shown in graphs. Statistical significance was calculated by student t-test; asterisks indicate * p-value < 0.05 and ** p-value < 0.01. Error bars represent SEM. (C) Fibroblasts were infected with 1 MOI of WT or ΔmiR-US22 TB40/E GFP virus and serum starved overnight before treating with 50 ng/mL of EGF. Samples were collected at 3 and 4 dpi, then separated on a SDS-PAGE gel, and blotted for α-EGR1, α- UL138 , α-IE1, and α-GAPDH. Normalized values for EGR1 and UL138 protein are below each band. Blot is representative of two independent experiments.

Article Snippet: EGR1 (44D5) , Rabbit , Cell Signaling , Western 1:1,000; ChIP 10 μL/ 4x10 6 cells.

Techniques: Transduction, Plasmid Preparation, Infection, SDS Page, Transfection

Journal: PLoS Pathogens

Article Title: Host signaling and EGR1 transcriptional control of human cytomegalovirus replication and latency

doi: 10.1371/journal.ppat.1008037

Figure Lengend Snippet: (A) CD34 + HPCs were infected with TB40/E GFP (MOI = 2). At 2 and 6 dpi, mRNA libraries were prepared for Illumina sequencing. Relative expression of EGR1, EGR2, EGR3, and WT1 was calculated by fragments per kilobase per million reads (FPKM) and normalized to EGR1 2 dpi levels. Error bars represent the range of gene expression between two independent donors. (B) Fibroblasts were infected with WT TB40/E GFP (MOI = 1) and RNA was isolated at 0–72 hpi. EGR1 mRNA was quantified relative to H6PD by RT-qPCR. Results from 3 independent replicates are graphed error bars represent SEM. Statistical significance was calculated by One-Way ANOVA with Tukey’s correction and represented by an asterisk (* p-value < 0.05). (C) Fibroblasts were infected with TB40/E GFP (MOI = 1) and transferred to serum-free media at 24 hpi. At 48 hpi, samples were pulsed with 10 nM of EGF for 1 h. Lysates were separated by SDS-PAGE and immunoblotted with α-EGR1, α-IE1/2, and α-tubulin. EGR1 protein levels were normalized to the uninfected sample stimulated with EGF and the mean from 3 independent replicates is graphed. Error bars represent SEM. We calculated statistical significance by two-way ANOVA with Tukey’s correction and represented significance by asterisks (* p-value < 0.05; **** p-value < 0.0001).

Article Snippet: EGR1 (44D5) , Rabbit , Cell Signaling , Western 1:1,000; ChIP 10 μL/ 4x10 6 cells.

Techniques: Infection, Sequencing, Expressing, Isolation, Quantitative RT-PCR, SDS Page

Journal: PLoS Pathogens

Article Title: Host signaling and EGR1 transcriptional control of human cytomegalovirus replication and latency

doi: 10.1371/journal.ppat.1008037

Figure Lengend Snippet: (A) HEK293T cells were co-transfected with either empty vector (minus sign) or EGR1 3xFLAG and a promoterless plasmid containing UL133/8 sequences or UL133/8 where EGR1 sites were mutated in combination (ΔSite1+2) or individually (ΔSite 1 or ΔSite 2). At 48 h, lysates were separated by SDS-PAGE, and proteins detected using α-Flag, α-UL138, and α-tubulin. UL138 protein levels in EGR1 3xFLAG transfections were normalized to control levels to determine UL138 induction. The results from 4 independent replicates are graphed. Statistical significance was calculated by One-Way ANOVA with Bonferroni correction (* p-value < 0.05 and ** p-value < 0.01). (B) HEK293T cells were co-transfected with the UL133/8 vector or the UL133/8 vector where EGR1 sites (ΔSite1, ΔSite 2) were disrupted and negative control siRNA, EGR1 siRNA, or miR-US22. Cells were transferred to serum-free media at 24 h. At 48 h, samples were stimulated with 50 ng/mL EGF for 1h and then lysed, separated by SDS-PAGE, and proteins detected using α-UL138 and α-GAPDH. UL138 levels are normalized to negative control. A representative blot of 2 independent experiments is shown.

Article Snippet: EGR1 (44D5) , Rabbit , Cell Signaling , Western 1:1,000; ChIP 10 μL/ 4x10 6 cells.

Techniques: Transfection, Plasmid Preparation, SDS Page, Negative Control

Journal: PLoS Pathogens

Article Title: Host signaling and EGR1 transcriptional control of human cytomegalovirus replication and latency

doi: 10.1371/journal.ppat.1008037

Figure Lengend Snippet: (A) Fibroblasts were infected with WT TB40/E GFP or EGR1 binding mutant viruses, ΔSite 1 or ΔSite 2 (MOI = 0.02). Cells and media were collected from 0–16 dpi and virus titers measured by TCID 50 . The average of 3 independent replicate experiments is shown. (B) Fibroblasts were infected with WT or EGR1 binding mutant viruses. Samples were lysed at 48 hpi, separated by SDS-PAGE and proteins detected using α-IE1/2, α-UL135, and α-UL138, and α-tubulin. UL138 protein levels were quantified and each mutant was normalized to WT over 3 independent experiments. The average value is graphed with error bars representing SEM and statistical significance is calculated by One-way ANOVA with Bonferroni correction (* p-value <0.05). (C) Fibroblasts were infected with WT TB40/E GFP or EGR1 binding mutant viruses, ΔSite 1 or ΔSite 2, (MOI = 1) and transferred to serum-free media at 24 hpi. At 48 hpi, samples were pulsed with 10 nM EGF for 1h and processed for ChIP-qPCR using SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling). The presence of EGR1 Site 1 sequence was calculated relative to a 2% input control and normalized to WT levels.

Article Snippet: EGR1 (44D5) , Rabbit , Cell Signaling , Western 1:1,000; ChIP 10 μL/ 4x10 6 cells.

Techniques: Infection, Binding Assay, Mutagenesis, SDS Page, Chromatin Immunoprecipitation, Sequencing

Journal: PLoS Pathogens

Article Title: Host signaling and EGR1 transcriptional control of human cytomegalovirus replication and latency

doi: 10.1371/journal.ppat.1008037

Figure Lengend Snippet: Our data demonstrates that EGFR signaling promotes EGR-1 expression through MEK/ERK signaling pathways. EGR-1 stimulates UL138 expression and UL138 feeds back to sustain EGFR signaling. UL135 targets EGFR for turnover and CMV miR-US22 targets EGR1 to negatively regulate this cycle for reactivation.

Article Snippet: EGR1 (44D5) , Rabbit , Cell Signaling , Western 1:1,000; ChIP 10 μL/ 4x10 6 cells.

Techniques: Expressing

Journal: PLoS Pathogens

Article Title: Host signaling and EGR1 transcriptional control of human cytomegalovirus replication and latency

doi: 10.1371/journal.ppat.1008037

Figure Lengend Snippet: Primer sequences.

Article Snippet: EGR1 (44D5) , Rabbit , Cell Signaling , Western 1:1,000; ChIP 10 μL/ 4x10 6 cells.

Techniques: Sequencing

Journal: PLoS Pathogens

Article Title: Host signaling and EGR1 transcriptional control of human cytomegalovirus replication and latency

doi: 10.1371/journal.ppat.1008037

Figure Lengend Snippet: Antibody description and sources.

Article Snippet: EGR1 (44D5) , Rabbit , Cell Signaling , Western 1:1,000; ChIP 10 μL/ 4x10 6 cells.

Techniques: Concentration Assay, Western Blot

Journal: The international journal of biochemistry & cell biology

Article Title: Cellular steatosis in ethanol oxidizing-HepG2 cells is partially controlled by the transcription factor, early growth response-1

doi: 10.1016/j.biocel.2012.10.002

Figure Lengend Snippet: (A) Egr-1 promoter activity performed in VL-17A cells exposed to zero (controls) or 50mM ethanol for various time periods as indicated in the figure. (B) Egr-1 mRNA levels in HepG2 and VL-17A cells exposed to zero (controls) or 50 mM ethanol for the indicated time periods. At each time point the control value (i.e. cells treated without ethanol) is 1 for both cell-types and is not shown (C) Densitometric ratios of Egr-1 to β-actin in VL-17A cells exposed to zero (controls) or 50 mM ethanol for various time points as indicated in the figure. (D) Densitometric ratios of nuclear Egr-1 to β-actin in VL-17A cells exposed to zero (controls) or 50mM ethanol for the indicated times. (E) Densitometric ratios of Egr-1 to β-actin in VL-17A cells treated with ethanol in the presence and absence of 4- MP. Data are mean values ± SEM of 4 to 8 samples. ****P < 0.0001, ***P < 0.001,**P < 0.01, *P< 0.05 (control vs ethanol). # vs †, P =0.03 (3 hr ethanol vs 6 hr ethanol), # vs ¶, P =0.006 (3 hr ethanol vs 24 hr ethanol), † vs ¶, P =0.04 (6 hr ethanol vs 24 hr ethanol).

Article Snippet: 2.1.1:Reagents Anti-Egr-1 was obtained from Cell Signaling Technology Inc. (Danvers, MA).

Techniques: Activity Assay

Journal: The international journal of biochemistry & cell biology

Article Title: Cellular steatosis in ethanol oxidizing-HepG2 cells is partially controlled by the transcription factor, early growth response-1

doi: 10.1016/j.biocel.2012.10.002

Figure Lengend Snippet: >(A) Residual ethanol and acetaldehyde in culture medium after 50mM ethanol treatment for the indicated times. (B) Representative western blot showing Egr-1 protein and densitometric ratios of Egr-1 to β-actin in VL-17A cells exposed to the indicated ethanol concentrations for 24 hr. Data are mean values ± SEM of 4 to 8 samples per group. ****P < 0.0001, ***P < 0.001,**P < 0.01, *P < 0.05 (control vs ethanol). # vs †, P =0.02; # vs ¶, P =0.006; # vs ║, P =0.0009; † vs ¶, P =0.007; † vs║, P =0.001; ¶ vs║, P =0.02.

Article Snippet: 2.1.1:Reagents Anti-Egr-1 was obtained from Cell Signaling Technology Inc. (Danvers, MA).

Techniques: Western Blot

Journal: The international journal of biochemistry & cell biology

Article Title: Cellular steatosis in ethanol oxidizing-HepG2 cells is partially controlled by the transcription factor, early growth response-1

doi: 10.1016/j.biocel.2012.10.002

Figure Lengend Snippet: Densitometric ratios of Egr-1 to β-actin in HepG2, VA-13, E-47 and VL-17A cells treated with (+) and without ((−) controls) 50 mM ethanol for 24 hr. Data are mean values of 4 samples ± SEM. ****P < 0.0001, ***P < 0.001,**P < 0.01, *P < 0.05 (control vs ethanol).

Article Snippet: 2.1.1:Reagents Anti-Egr-1 was obtained from Cell Signaling Technology Inc. (Danvers, MA).

Techniques:

Journal: The international journal of biochemistry & cell biology

Article Title: Cellular steatosis in ethanol oxidizing-HepG2 cells is partially controlled by the transcription factor, early growth response-1

doi: 10.1016/j.biocel.2012.10.002

Figure Lengend Snippet: (A) Egr-1 promoter activity performed in HepG2 cells treated or untreated with two acetaldehyde concentrations (100 and 300uM) or 50 mM ethanol for 1 hr. (B) Representative western blot showing Egr-1 protein and densitometric ratios of Egr-1 to β- actin in HepG2 cells exposed to 300 uM acetaldehyde for 1 and 3 hr. ****P < 0.0001, ***P < 0.001,**P < 0.01, *P < 0.05 (control vs ethanol).

Article Snippet: 2.1.1:Reagents Anti-Egr-1 was obtained from Cell Signaling Technology Inc. (Danvers, MA).

Techniques: Activity Assay, Western Blot

Journal: The international journal of biochemistry & cell biology

Article Title: Cellular steatosis in ethanol oxidizing-HepG2 cells is partially controlled by the transcription factor, early growth response-1

doi: 10.1016/j.biocel.2012.10.002

Figure Lengend Snippet: (A) Egr-1 protein levels after adenovirus mediated Egr-1 over-expression. # vs †, P =0.03; # vs ¶, P =0.01; † vs ¶, P =0.02; † vs║, P =0.03; ¶ vs║, P =0.01. (B) Triglyceride levels in un-transduced, Adv-Egr-1-transduced and Adv-GFP transduced cells exposed to zero (controls) or 50 mM EtOH for 24 hr. # vs †, P =0.02; † vs ¶, P =0.01; † vs║, P =0.04 (C) Egr-1 protein levels after siRNA mediated Egr-1 knockdown in VL-17A cells. # vs †, P =0.003; # vs║, P =0.03; † vs ¶, P =0.006; † vs║, P=0.01; ¶ vs║, P =0.03. (D) Triglyceride levels in untransfected, scrambled siRNA transfected, Egr-1siRNA transfected cells exposed to zero (controls) or 50 mM EtOH for 24 hr. # vs ¶, P =0.04; † vs ¶, P =0.04. Data are mean values of 6 to 8 samples ± SEM. ****P < 0.0001, ***P < 0.001,**P < 0.01, *P < 0.05 (control vs ethanol).

Article Snippet: 2.1.1:Reagents Anti-Egr-1 was obtained from Cell Signaling Technology Inc. (Danvers, MA).

Techniques: Over Expression, Transfection

Journal: The international journal of biochemistry & cell biology

Article Title: Cellular steatosis in ethanol oxidizing-HepG2 cells is partially controlled by the transcription factor, early growth response-1

doi: 10.1016/j.biocel.2012.10.002

Figure Lengend Snippet: (A) Representative western blot and mRNA levels of Egr-1 in scrambled siRNA- and Egr-1 siRNA-transfected cells treated with and without (controls) 50 mM ethanol for 3 and 6 h. Three hours: # vs. †, P = 0.0001; # vs. ¶, P = 0.0009. Six hours: # vs. †, P = 0.0007; # vs. ¶, P = 0.0007. (B) SREBP1c mRNA levels in scrambled siRNA- and Egr-1siRNA-transfected cells exposed to zero (controls) or 50 mM EtOH for 3 and 6 h. Three hours: # vs. †, P = 0.02. Six hours: # vs. †, P = 0.008 (C). TNF-α mRNA levels in scrambled siRNA- and Egr-1siRNA-transfected cells exposed to zero (controls) or 50 mM EtOH for 3 and 6 h. Three hours: # vs. †, P = 0.04. Six hours: # vs. †, P = 0.01. Data are mean values of 5 samples ± SEM. ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05 (control vs. ethanol).

Article Snippet: 2.1.1:Reagents Anti-Egr-1 was obtained from Cell Signaling Technology Inc. (Danvers, MA).

Techniques: Western Blot, Transfection

Journal: The international journal of biochemistry & cell biology

Article Title: Cellular steatosis in ethanol oxidizing-HepG2 cells is partially controlled by the transcription factor, early growth response-1

doi: 10.1016/j.biocel.2012.10.002

Figure Lengend Snippet: (A) Egr-1 mRNA levels in scrambled siRNA transfected, Egr-1 and SREBP1c siRNA transfected cells exposed to zero (controls) or 50 mM EtOH for 24 hr in VL-17A cells. # vs †, P =0.0001; # vs ¶, P =0.0001. (B) SREBP1c mRNA levels in scrambled siRNA transfected, Egr-1and SREBP1c siRNA transfected cells exposed to zero (controls) or 50 mM EtOH for 24 hr in VL-17A cells. # vs †, P =0.002; # vs ¶, P =0.001. (C) Triglyceride levels in scrambled siRNA transfected, Egr-1and SREBP1c siRNA transfected cells exposed to zero (controls) or 50 mM EtOH for 24 hr in VL-17A cells. Data are mean values of 5 samples ± SEM. # vs †, P =0.00003. ****P < 0.0001, ***P < 0.001,**P < 0.01, *P < 0.05 (control vs ethanol).

Article Snippet: 2.1.1:Reagents Anti-Egr-1 was obtained from Cell Signaling Technology Inc. (Danvers, MA).

Techniques: Transfection

Journal: The international journal of biochemistry & cell biology

Article Title: Cellular steatosis in ethanol oxidizing-HepG2 cells is partially controlled by the transcription factor, early growth response-1

doi: 10.1016/j.biocel.2012.10.002

Figure Lengend Snippet: ADH-catalysis of ethanol oxidation produces acetaldehyde, which enhances Egr-1 gene transcription by activating Egr-1 promoter. Increased gene transcription enhances Egr-1 mRNA which precedes a rise in nuclear Egr-1 protein. CYP2E1 and ADH catalysis of ethanol oxidation generates reactive products and inhibits proteasome activity. Such inhibition stabilizes Egr-1 protein, from hydrolysis by the proteasome. Finally, Egr-1 regulates SREBP1c and TNF-α to initiate ethanol induced steatosis

Article Snippet: 2.1.1:Reagents Anti-Egr-1 was obtained from Cell Signaling Technology Inc. (Danvers, MA).

Techniques: Activity Assay, Inhibition