Journal: Oncotarget

Article Title: Loss of Egr1, a human del5q gene, accelerates BCR-ABL driven chronic myelogenous leukemia

doi: 10.18632/oncotarget.20612

Figure Lengend Snippet: A. Kaplan-Meier survival curves for recipients of MIGR1 or MIG BCR-ABL-transduced BM cells from WT or Egr1 -/- donor mice. WT recipient mice were transplanted with BCR-ABL-expressing BM or MIGR1-expressing BM. Following transplantation, animals were observed for signs of disease and euthanized at first signs of morbidity. Statistical analysis used was Log-rank (mantel-Cox) test, P value < 0.0001. Egr1 -/- /BCR-ABL ( n = 8) median survival 24.5 days, WT/BCR-ABL ( n = 9) median survival 35 days. WT MIG ( n = 4) and Egr1 -/- MIG ( n = 4). Animals transplanted with MIGR1-infected BM did not show any signs of leukemia and were euthanized at the end of the experiments. B. Most leukemic mice exhibited myeloid disease. GFP+ BM cells harvested from leukemic mice when they exhibited signs of leukemia were prepared for antibody treatment, and incubated with anti-mouse Gr-1 and B220 antibodies. Following Facs, data analysis was performed using FloJo software. (Although all gates were not drawn at the 10 2 positions on the y-axis, the cell populations are clearly observed and this does not impede the interpretation of the data). Eight to ten mice were analyzed from each genotype, and most of them (75-80%) developed myeloid leukemia. C. Representatives spleens of mice transplanted with either WT/BCR-ABL or Egr1 -/- /BCR-ABL BM cells at 18 days post-BMT. Spleens were removed from mice transplanted with either WT/BCR-ABL or Egr1 -/- /BCR-ABL BM cells following euthanasia at 18 days post-BMT.

Article Snippet: Probes used were the following, all purchased from Life Technologies: Mm00656724_m1 (mouse Egr1 ), and Mm04277571_S1, for 18, which was used as endogenous control.

Techniques: Expressing, Transplantation Assay, Infection, Incubation, Software

Journal: Oncotarget

Article Title: Loss of Egr1, a human del5q gene, accelerates BCR-ABL driven chronic myelogenous leukemia

doi: 10.18632/oncotarget.20612

Figure Lengend Snippet: Lethally irradiated WT recipient mice were transplanted with BM from WT or Egr1 -/- mice infected with BCR-ABL-containing retrovirus as detailed above. For panels A. , B. and C. , mice were euthanized when moribund. Facs analysis was done on peripheral blood (A), BM (B) and spleen cells (C) to assess % GFP + cells. (A), P value = 0.0442; (B), P value = 0.0370; (C), P value = 0.0442. WT/BCR-ABL n = 6, Egr1 -/- /BCR-ABL n = 5. D. & E. Mice were euthanized 20 days after BMT, and spleen, liver and total body weight were determined. Mice transplanted with MIGR1-infected BM were used as controls. Values plotted represent the ratio of spleen or liver weight to total body weight (TBW). (D) Analysis of spleen weight. Spleens of Egr1 -/- /BCR-ABL -transplanted mice were significantly larger than in controls and in WT/BCR-ABL-transplanted mice. %TBW spleen: Egr1 -/- /BCR-ABL vs. WT/BCR-ABL- p value = 0.008; WT/BCR-ABL vs. Control - p value = 0.0075; Egr1 -/- /BCR-ABL vs. Control - p value = 0.0002. (E) Analysis of liver weight. Livers of Egr1 -/- /BCR-ABL -transplanted mice were significantly larger than those of animals transplanted with WT/BCR-ABL BM cells. %TBW liver: Egr1 -/- /BCR-ABL vs. WT/BCR-ABL - significant, p value = 0.0080; WT/BCR-ABL vs. Control - not significant, p value = 0.4163; Egr1 -/- /BCR-ABL vs. Control - Significant, p value = 0.0053. Control ( n = 3), WT/BCR-ABL ( n = 5), Egr1 -/- /BCR-ABL ( n = 5); F. H&E stained slides obtained from organs of leukemic mice showed more extensive infiltration of leukemic cells in Egr1 -/- /BCR-ABL BM-transplanted mice compared to WT counterparts, in spleen and lungs. No clear infiltration of leukemic cells in the livers in either of the genotypes was observed. Five slides from organs from 3 mice for each genotype were examined; photograph is representative of what was observed for each genotype. Mice were displaying signs of leukemia when euthanized. Arrows indicate infiltration of leukemic cells. (10 × magnification). G. Summary of survival curves. Kaplan-Meier survival curves for recipients of MIGR1 or MIG BCR-ABL-transduced BM cells from WT or Egr1 -/- donor mice.

Article Snippet: Probes used were the following, all purchased from Life Technologies: Mm00656724_m1 (mouse Egr1 ), and Mm04277571_S1, for 18, which was used as endogenous control.

Techniques: Irradiation, Infection, Control, Staining

Journal: Oncotarget

Article Title: Loss of Egr1, a human del5q gene, accelerates BCR-ABL driven chronic myelogenous leukemia

doi: 10.18632/oncotarget.20612

Figure Lengend Snippet: A. WT BM from 5-FU treated mice was transduced with either MSCV-BCR-ABL-IRES-GFP (MIG BCR-ABL) or the MSCV-IRES-GFP (MIGR1) retroviral vector. GFP + cells were selected by cell sorting, expanded, and RNA extracted. Total RNA was analyzed by real time PCR for Egr1 expression using taqman probe. 18S rRNA probe was used as an internal control. Each sample comes from a different mouse. WT/MIG n = 4, WT/BCR-ABL n = 5. B. Spleens were harvested from mice transplanted with WT BM infected with either MIGR1 vector (control) or MIG BCR-ABL vector, 20 days post-BMT. Cells from whole spleens were dissociated and red blood cells removed prior to RNA extraction. WT/MIG n = 3, WT/BCR-ABL n = 8.

Article Snippet: Probes used were the following, all purchased from Life Technologies: Mm00656724_m1 (mouse Egr1 ), and Mm04277571_S1, for 18, which was used as endogenous control.

Techniques: Transduction, Retroviral, Plasmid Preparation, FACS, Real-time Polymerase Chain Reaction, Expressing, Control, Infection, RNA Extraction

Journal: Oncotarget

Article Title: Loss of Egr1, a human del5q gene, accelerates BCR-ABL driven chronic myelogenous leukemia

doi: 10.18632/oncotarget.20612

Figure Lengend Snippet: For A. and B. , Egr1 -/- and WT BM cells were infected with BCR-ABL expressing vectors, and GFP+ cells were sorted and used for analysis. Viability was assessed using MTS assay by normalizing to day 0. BCR-ABL infected Egr1 -/- BM cells showed enhanced viability at the three time-points compared to BCR-ABL infected WT BM cells. Day 1 - P value < 0.0001; Day 2 - P value = 0.0003; Day 3 - P value = 0.0013. Data is representative of five independent experiments. (B) GFP+ sorted BM cells were incubated with Annexin V antibody and PI, and Facs analysis was done. BCR-ABL infected Egr1 -/- BM have fewer Annexin V positive cells, thus fewer apoptotic cells, than BCR-ABL infected WT BM, P value = 0.033. Represented here are the percentage of early (Annexin V + only) and late (Annexin V + and PI + ) apoptosis. Data is representative of 3 independent experiments. C. 20 days following BMT, mice were injected intraperitoneally with BrdU and euthanized after two hours. BM was harvested and prepared for cell proliferation assessment using BrdUassay. P value = 0.01. WT/BCR-ABL: G1-55.3%, S-8.3%, G2-M-11.1%; Egr1 -/- /BCR-ABL: G1-60.3%, S-15.2%, G2-M-12.9%. Data is representative of 3 independent experiments. WT/BCR-ABL n = 3, Egr1 -/- /BCR-ABL n = 4.

Article Snippet: Probes used were the following, all purchased from Life Technologies: Mm00656724_m1 (mouse Egr1 ), and Mm04277571_S1, for 18, which was used as endogenous control.

Techniques: Infection, Expressing, MTS Assay, Incubation, Injection

Journal: Oncotarget

Article Title: Loss of Egr1, a human del5q gene, accelerates BCR-ABL driven chronic myelogenous leukemia

doi: 10.18632/oncotarget.20612

Figure Lengend Snippet: A.-B. The leukemia stem cells are enriched in BM from mice transplanted with Egr1 -/- /BCR-ABL BM compared to WT/BCR-ABL BM. BM was harvested 20 days post BMT. Representative Facs data of GFP + Lin - cells from one of the mice analyzed for each genotype. P value = 0.0156. ( n = 3 for each genotype) C. BM from primary transplanted mice was harvested on day 14 post BMT and red blood cells were lysed. 1x10 6 BM cells from primary transplanted mice were injected into lethally irradiated secondary recipient mice, and Kaplan-Meier survival curves for recipients are shown. Statistical analysis was performed using Log-Rank (Mantel-Cox) test. P value = 0.0028. Median Survival: WT/BCR-ABL 32 days; Egr1 -/- /BCR-ABL 11 days. WT/BCR-ABL ( n = 6) and Egr1 -/- /BCR-ABL ( n = 7).

Article Snippet: Probes used were the following, all purchased from Life Technologies: Mm00656724_m1 (mouse Egr1 ), and Mm04277571_S1, for 18, which was used as endogenous control.

Techniques: Injection, Irradiation

Journal: Oncotarget

Article Title: Loss of Egr1, a human del5q gene, accelerates BCR-ABL driven chronic myelogenous leukemia

doi: 10.18632/oncotarget.20612

Figure Lengend Snippet: GFP + cells (WT/BCR-ABL and Egr1 -/- /BCR-ABL) were plated for colonies, 8-12 days later colonies were counted and plated for the next round of colonies. A. Data from one representative experiment (experiment 2 shown in Table). Statistical analysis: first round, P value = 0.3232 not significant; second round: P value = 0.0008, third round: P value < 0.0001; fourth round: P value < 0.0001. Colony number is average number of colonies obtained from three 35mm dishes. B. Photomicrograph of representative plate from 4 th round of plating, 4 × magnification.

Article Snippet: Probes used were the following, all purchased from Life Technologies: Mm00656724_m1 (mouse Egr1 ), and Mm04277571_S1, for 18, which was used as endogenous control.

Techniques:

Journal: Oncology Reports

Article Title: Lentinan induces apoptosis of mouse hepatocellular carcinoma cells through the EGR1/PTEN/AKT signaling axis

doi: 10.3892/or.2023.8579

Figure Lengend Snippet: Effects of LNT on the EGR1/PTEN/AKT axis in Hepa1-6 cells. (A) Protein expression levels of EGR1, PTEN, p-Akt and phosphorylation of Akt after treatment with a gradient of LNT concentrations as detected by WB. All data for protein expression were normalized using β-actin as a loading reference. (B) Immunofluorescence co-staining was used to assess the localization and expression of EGR1 and PTEN in Hepa1-6 cells treated with a gradient of LNT concentrations. Images were observed at ×630 magnification. Scale bars, 50 µm (white). (C) WB was used to detect the expression of EGR1 in the nuclear and cytoplasmic fractions following treatment with a gradient of LNT concentrations. (D) WB was used to detect the expression of EGR1, PTEN, p-Akt, and phosphorylation of Akt after EGR1 overexpression. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. LNT, Lentinan; EGR1, early growth response 1; p-, phosphorylated; PARP1, poly-(ADP ribose) polymerase 1; Hsp60, heat shock protein 60; OE, overexpression; WB, western blotting.

Article Snippet: Subsequently, they were incubated with a mixture of different primary antibodies, including mouse EGR1 (1:100; cat. no. H00001958-M03; Novus Biologicals, LLC) and rabbit PTEN (1:100; cat. no. AF6351; Affinity Biosciences), overnight at 4°C.

Techniques: Expressing, Phospho-proteomics, Immunofluorescence, Staining, Over Expression, Western Blot

Journal: Oncology Reports

Article Title: Lentinan induces apoptosis of mouse hepatocellular carcinoma cells through the EGR1/PTEN/AKT signaling axis

doi: 10.3892/or.2023.8579

Figure Lengend Snippet: Inhibitory effect of LNT on DEN-induced primary liver cancer in mice. (A) The expression levels of EGR1, PTEN and other proteins in the liver tissues of the normal group and the model group, as analyzed by WB. (B) Expression of EGR1, PTEN and other proteins in liver tissues of the model and LNT-treated groups, as analyzed by WB. (C) Immunohistochemical analysis of EGR1, PTEN and Ki-67 in the tissue sections. Magnification, ×400. Scale bars, 200 µm (black). *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. LNT, Lentinan; DEN, diethylnitrosamine; EGR1, early growth response 1; WB, western blotting; p-, phosphorylated; PCNA, proliferating cell nuclear antigen.

Article Snippet: Subsequently, they were incubated with a mixture of different primary antibodies, including mouse EGR1 (1:100; cat. no. H00001958-M03; Novus Biologicals, LLC) and rabbit PTEN (1:100; cat. no. AF6351; Affinity Biosciences), overnight at 4°C.

Techniques: Expressing, Immunohistochemical staining, Western Blot

Journal: Oncology Reports

Article Title: Lentinan induces apoptosis of mouse hepatocellular carcinoma cells through the EGR1/PTEN/AKT signaling axis

doi: 10.3892/or.2023.8579

Figure Lengend Snippet: Possible mechanism of LNT against liver cancer through the EGR1/PTEN/AKT axis. Blunt arrows indicate that the signaling pathways are inhibited, whilst pointed arrows indicate that the signaling pathways are activated. LNT, Lentinan; EGR1, early growth response 1.

Article Snippet: Subsequently, they were incubated with a mixture of different primary antibodies, including mouse EGR1 (1:100; cat. no. H00001958-M03; Novus Biologicals, LLC) and rabbit PTEN (1:100; cat. no. AF6351; Affinity Biosciences), overnight at 4°C.

Techniques: Protein-Protein interactions

Journal: Cell Death and Differentiation

Article Title: NFκB mitigates the pathological effects of misfolded α1-antitrypsin by activating autophagy and an integrated program of proteostasis mechanisms

doi: 10.1038/s41418-018-0130-7

Figure Lengend Snippet: Activation of NFκB and downregulation of Egr1 in the liver of the PiZ mouse model. a Transcriptional changes in the Z mouse model with liver-specific inducible expression of ATZ compared to known downstream targets of NFκB. In the left column genes that are known downstream targets of NFκB are listed and the arrow indicates whether the expression of each gene is increased or decreased by the action of NFκB in conventional inflammatory states. In the right column results of RT-PCR show whether these genes in gene expression occur in the liver of the Z mouse with liver-specific inducible expression [2] when ATZ expression is induced by withdrawal of doxycycline from the drinking water (+ indicates that the change in expression is reproduced in the Z mouse liver when ATZ gene expression is induced; − indicates that there is no change in expression of the gene when ATZ gene expression is induced in the Z mouse). NS indicates no significant difference. b NFκB DNA-binding activity in PiZ and C57 (C57/BL6) mouse liver was assayed by electrophoretic mobility shift assay and the specific gel shift band was analyzed by densitometric scanning. Results are shown as mean ± SE for n = 3 mice each, p < 05. Asterisk indicates statistical significance from C57. c Chromatin immunoprecipitation assay in PiZ and C57 (C57/BL6) liver using antibodies to p50, p65, and non-specific IgG. c Expression of Egr1 in PiZ and C57 (C57/BL6) liver by RT-PCR. d Expression of Egr1 in PiZ and control (FVB/N) liver by QPCR. Results are shown as mean ± SE for n = 3 mice each, p < 0.01. Asterisk indicates statistical significance from control

Article Snippet: Detection of human ATZ, mouse EGR1, Collagens (1, 3, and 5), Acta 2, MMPs (7, 9, and 12), p62, ATG5, ATG7, ATG12, Beclin 1, and human EGR1, MMPs (7 and 12) were achieved using specific Taqman Gene Expression Assays (Hs01097800_m1, Mm00656724_m1, Mm00801666_m1, Mm00802331_m1, Mm00483675_m1, Mm00487724_m1, Mm00442991_m1, Mm00500554_m1, Mm01187303_m1, Mm01187303_m1, Mm00512209_m1, Mm00503201_m1, Mm01265461_m1, Hs00152928_m1, and Hs01042796_m1, Hs00159178_m1, respectively).

Techniques: Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Binding Assay, Activity Assay, Electrophoretic Mobility Shift Assay, Gel Shift, Chromatin Immunoprecipitation, Control

Journal: Cell Death and Differentiation

Article Title: NFκB mitigates the pathological effects of misfolded α1-antitrypsin by activating autophagy and an integrated program of proteostasis mechanisms

doi: 10.1038/s41418-018-0130-7

Figure Lengend Snippet: Activation of NFκB and downregulation of Egr1 in the HTO/Z cell line model. a NFκB DNA-binding activity in HTO/Z and control HTO/M cell line was assayed by electrophoretic mobility shift assay and the specific gel shift band was analyzed by densitometric scanning. Results are shown as mean ± SE for n = 3 biological replicates each, p < 01. b Immunoblot analysis for steady-state levels of ATZ, Egr1, and GAPDH in the HTO/Z cell line in the presence or absence of dox with relative densitometic intensity for Egr1 shown on the right in mean ± SE, p < 0.05. c Immunoblot analysis for steady-state levels of Egr1 in HTO/M and HTO/Z cell line in the absence of dox. Gelcode blue staining was used as a loading control. Results of densitometric scanning is shown at the right of b and c as mean ± SE, p < 0.05 for n = 3 biological replicates. Asterisk indicates statistical significance from control

Article Snippet: Detection of human ATZ, mouse EGR1, Collagens (1, 3, and 5), Acta 2, MMPs (7, 9, and 12), p62, ATG5, ATG7, ATG12, Beclin 1, and human EGR1, MMPs (7 and 12) were achieved using specific Taqman Gene Expression Assays (Hs01097800_m1, Mm00656724_m1, Mm00801666_m1, Mm00802331_m1, Mm00483675_m1, Mm00487724_m1, Mm00442991_m1, Mm00500554_m1, Mm01187303_m1, Mm01187303_m1, Mm00512209_m1, Mm00503201_m1, Mm01265461_m1, Hs00152928_m1, and Hs01042796_m1, Hs00159178_m1, respectively).

Techniques: Activation Assay, Binding Assay, Activity Assay, Control, Electrophoretic Mobility Shift Assay, Gel Shift, Western Blot, Staining

Journal: Cell Death and Differentiation

Article Title: NFκB mitigates the pathological effects of misfolded α1-antitrypsin by activating autophagy and an integrated program of proteostasis mechanisms

doi: 10.1038/s41418-018-0130-7

Figure Lengend Snippet: Expression of Egr1 in PiZ × p50-null mouse liver. a QPCR. Results are shown as mean ± SE for n = 3 biological replicates each. Single asterisk indicates statistical significance for PiZ versus p50-null (p < 0.01) and double asterisk indicates statistical significance for PiZ × p50-null versus PiZ (p < 0.05). b Immunoblot analysis with densitometric scanning results at right. Gelcode blue staining is used as loading control. x indicates statistical significance for PiZ versus p50-null (p < 0.01) and double asterisk indicates statistical significance for PiZ versus PiZ × p50-null (p < 0.01) for n = 4 biological replicates

Article Snippet: Detection of human ATZ, mouse EGR1, Collagens (1, 3, and 5), Acta 2, MMPs (7, 9, and 12), p62, ATG5, ATG7, ATG12, Beclin 1, and human EGR1, MMPs (7 and 12) were achieved using specific Taqman Gene Expression Assays (Hs01097800_m1, Mm00656724_m1, Mm00801666_m1, Mm00802331_m1, Mm00483675_m1, Mm00487724_m1, Mm00442991_m1, Mm00500554_m1, Mm01187303_m1, Mm01187303_m1, Mm00512209_m1, Mm00503201_m1, Mm01265461_m1, Hs00152928_m1, and Hs01042796_m1, Hs00159178_m1, respectively).

Techniques: Expressing, Western Blot, Staining, Control

Journal: Cell Death and Differentiation

Article Title: NFκB mitigates the pathological effects of misfolded α1-antitrypsin by activating autophagy and an integrated program of proteostasis mechanisms

doi: 10.1038/s41418-018-0130-7

Figure Lengend Snippet: Expression of NFκB target genes Egr1, MMP7, and MMP12 in livers from ATD patients (n = 5) and normal controls (n = 7) by qRT-PCR. Results are shown as fold change (mean ± SE). Asterisk indicates statistically significant difference

Article Snippet: Detection of human ATZ, mouse EGR1, Collagens (1, 3, and 5), Acta 2, MMPs (7, 9, and 12), p62, ATG5, ATG7, ATG12, Beclin 1, and human EGR1, MMPs (7 and 12) were achieved using specific Taqman Gene Expression Assays (Hs01097800_m1, Mm00656724_m1, Mm00801666_m1, Mm00802331_m1, Mm00483675_m1, Mm00487724_m1, Mm00442991_m1, Mm00500554_m1, Mm01187303_m1, Mm01187303_m1, Mm00512209_m1, Mm00503201_m1, Mm01265461_m1, Hs00152928_m1, and Hs01042796_m1, Hs00159178_m1, respectively).

Techniques: Expressing, Quantitative RT-PCR

Journal: iScience

Article Title: A mechanism of cooling hot tumors: Lactate attenuates inflammation in dendritic cells

doi: 10.1016/j.isci.2021.103067

Figure Lengend Snippet:

Article Snippet: Plasmid: Egr1 , OriGene Technologies , MC217560.

Techniques: Control, Recombinant, Isolation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Cell Isolation, Reporter Assay, Mutagenesis, Chromatin Immunoprecipitation, DNA Purification, Staining, Lactate Assay, Microarray, Negative Control, Plasmid Preparation, Software, Transfection