egr1 overexpression  (Genechem)

Journal: Neurotherapeutics

Article Title: The EGR1/ZFP36 axis governs glycosphingolipid metabolic reprogramming in monocyte-derived macrophages in guillain-barré syndrome

doi: 10.1016/j.neurot.2026.e00895

Figure Lengend Snippet: Analysis of differentially expressed genes in the peripheral blood immune cell profile and disease-characteristic monocyte subsets of GBS patients. (a–b) Types and proportions of immune cells in the peripheral blood of GBS patients compared to healthy controls. The proportion of CD14 + monocytes was significantly increased in GBS patients. ( c –d) Types and proportions of monocyte subsets in the peripheral blood of GBS patients compared to healthy controls. CD163 + and IL1R2 + monocyte subsets were enriched in GBS patients. (e) Volcano plot of the top 5 (IL1R2, FKBP5, CD163, SAP30, and CXCR4) upregulated and top 5 (EGR1, FOSB, KLF4, CXCL8, and CCL3) downregulated differentially expressed genes in IL1R2 + monocytes from GBS patients' peripheral blood. (f) Top 20 B P enriched by GO analysis of differentially expressed genes in IL1R2 + monocytes. (g) PPI network of differentially expressed genes in IL1R2 + monocytes ranked by node score.

Article Snippet: Lentiviral vectors for EGR1 overexpression, EGR1 knockdown (sh-EGR1), and corresponding controls were constructed by GeneChem (Shanghai, China).

Techniques:

Journal: Neurotherapeutics

Article Title: The EGR1/ZFP36 axis governs glycosphingolipid metabolic reprogramming in monocyte-derived macrophages in guillain-barré syndrome

doi: 10.1016/j.neurot.2026.e00895

Figure Lengend Snippet: Analysis of DEGs in sciatic nerve transcriptome data of EAN rats. (a) Volcano plot of DEGs during the peak period of sciatic nerve in EAN rats and the top 5 DEGs with the lowest P -value. Egr1 was a significantly downregulated gene. (b) Heatmap of the expression levels of the top 5 upregulated and downregulated genes during the peak period of sciatic nerve in EAN rats in various samples, showing consistent downregulation of Cyp2c11 and Egr1 across all EAN samples. (c) Immunoinfiltration analysis of sciatic nerve ssGSEA in EAN rats, revealing significantly elevated infiltration of monocyte lineage cells. (d) Intersection Venn diagram of DEGs in GBS peripheral blood monocytes and EAN sciatic nerve, identifying 14 common genes. (e) The correlation between 14 DEGs in the sciatic nerve of EAN rats and infiltrating immune cells demonstrates that EGR1, FOS, ZFP36, and FOSB were negatively correlated with monocyte-macrophage infiltration.

Article Snippet: Lentiviral vectors for EGR1 overexpression, EGR1 knockdown (sh-EGR1), and corresponding controls were constructed by GeneChem (Shanghai, China).

Techniques: Expressing

Journal: Neurotherapeutics

Article Title: The EGR1/ZFP36 axis governs glycosphingolipid metabolic reprogramming in monocyte-derived macrophages in guillain-barré syndrome

doi: 10.1016/j.neurot.2026.e00895

Figure Lengend Snippet: The expression levels of EGR1 and ZFP36 in GBS patients, monocyte-derived macrophages, and EAN. (a) The level of EGR1 in the cerebrospinal fluid of normal (n = 25) and GBS patients (n = 48). EGR1 was significantly downregulated in GBS patients. (b) ROC curve of diagnostic efficacy of EGR1 level in cerebrospinal fluid for GBS (AUC = 0.921). ( c –d) qRT-PCR was employed to quantify the mRNA expression levels of iNOS, CD86, Arg1, CD163, EGR1, and ZFP36 in M0, M1, and M2 monocyte-derived macrophages (n = 3). EGR1 and ZFP36 were downregulated in M1 macrophages but upregulated in M2 macrophages. (e) WB was performed to analyze the protein expression of EGR1 and ZFP36 in M0 and M1 macrophages, confirming reduced expression in M1 macrophages (n = 3). (f) Immunofluorescence was employed to assess the expression and localization of EGR1 and ZFP36 in M1 macrophages, showing cytoplasmic localization and reduced fluorescence intensity (n = 3). (g–h) The expression levels of EGR1 and ZFP36 in M1 macrophages from the peripheral blood of EAN rats were confirmed by qRT-qPCR and WB analyses, further confirming their downregulation (n = 3). Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs HC/M0/Con group.

Article Snippet: Lentiviral vectors for EGR1 overexpression, EGR1 knockdown (sh-EGR1), and corresponding controls were constructed by GeneChem (Shanghai, China).

Techniques: Expressing, Derivative Assay, Diagnostic Assay, Quantitative RT-PCR, Immunofluorescence, Fluorescence

Journal: Neurotherapeutics

Article Title: The EGR1/ZFP36 axis governs glycosphingolipid metabolic reprogramming in monocyte-derived macrophages in guillain-barré syndrome

doi: 10.1016/j.neurot.2026.e00895

Figure Lengend Snippet: EGR1 specifically binds to ZFP36 and activates its transcription. (a) Venn diagram of the intersection between EGR1 downstream binding targets predicted based on four datasets from the TFBS database and DEGs of IL1R2 + monocytes in GBS, identifying ZFP36 and THBS1 as common targets. (b) WB detection of ZFP36 expression in M0 macrophages after overexpression and knockdown of EGR1, showing that EGR1 positively regulates ZFP36 expression (n = 3). (c) Schematic diagram showing the binding between EGR1 and ZFP36 promoter. (d). Enrichment of EGR1 binding to ZFP36 promoter was detected in M0 macrophages by ChIP-qPCR analysis, confirming specific binding of EGR1 to the ZFP36 promoter (n = 3). (e) M0 macrophages were co-transfected with ZFP36-WT or ZFP36-MUT reporter plasmids and OE-EGR1 or OE-NC plasmids; subsequently, luciferase reporter assays were performed to evaluate EGR1-mediated regulation of the ZFP36 promoter, demonstrating that EGR1 activates ZFP36 transcription through direct promoter binding (n = 3). Data are presented as mean ± SD. ∗∗∗ p < 0.001 vs ctrl/sh-NC/PMA/PMA + OE-EGR1/OE-NC + ZFP36-WT/OE-EGR1+ZFP36-WT.

Article Snippet: Lentiviral vectors for EGR1 overexpression, EGR1 knockdown (sh-EGR1), and corresponding controls were constructed by GeneChem (Shanghai, China).

Techniques: Binding Assay, Expressing, Over Expression, Knockdown, ChIP-qPCR, Transfection, Luciferase

Journal: Neurotherapeutics

Article Title: The EGR1/ZFP36 axis governs glycosphingolipid metabolic reprogramming in monocyte-derived macrophages in guillain-barré syndrome

doi: 10.1016/j.neurot.2026.e00895

Figure Lengend Snippet: Transcriptomic and metabolomic pathway enrichment analysis in EGR1-overexpressing and macrophages. (a–b) Top 20 KEGG pathways from GSEA of the ZFP36 gene set in transcriptomic data of EGR1-overexpression/knockdown macrophages. Glycosphingolipid metabolism pathways were commonly enriched in both datasets. ( c –d) Top 20 KEGG pathways from metabolomic data of EGR1-overexpression/knockdown macrophages. Sphingolipid metabolism showed positive enrichment upon EGR1 overexpression and negative enrichment upon EGR1 knockdown.

Article Snippet: Lentiviral vectors for EGR1 overexpression, EGR1 knockdown (sh-EGR1), and corresponding controls were constructed by GeneChem (Shanghai, China).

Techniques: Metabolomic, Over Expression, Knockdown

Journal: Neurotherapeutics

Article Title: The EGR1/ZFP36 axis governs glycosphingolipid metabolic reprogramming in monocyte-derived macrophages in guillain-barré syndrome

doi: 10.1016/j.neurot.2026.e00895

Figure Lengend Snippet: Enrichment patterns of sphingolipid metabolism pathway genes and metabolites in EGR1-overexpressing/knockdown macrophages. (a) Heatmap of gene expression in the sphingolipid metabolism pathway in EGR1-overexpressing and EGR1-knockdown macrophages, showing upregulation of 17 genes including HEXA and HEXB in the flag-EGR1 group. (b) KEGG network diagram integrating changes in genes and metabolites within the sphingolipid metabolism pathway in EGR1-overexpressing macrophages, demonstrating coordinated upregulation of HEXA, HEXB, and their metabolites globotriaosylceramide and lactosylceramide.

Article Snippet: Lentiviral vectors for EGR1 overexpression, EGR1 knockdown (sh-EGR1), and corresponding controls were constructed by GeneChem (Shanghai, China).

Techniques: Knockdown, Gene Expression

Journal: Neurotherapeutics

Article Title: The EGR1/ZFP36 axis governs glycosphingolipid metabolic reprogramming in monocyte-derived macrophages in guillain-barré syndrome

doi: 10.1016/j.neurot.2026.e00895

Figure Lengend Snippet: EGR1 targets ZFP36 to regulate glycosphingolipid metabolism and macrophage phenotype. (a) qRT-PCR was conducted to detect the efficiency of OE-ZFP36 and si-ZFP36 in macrophages, confirming successful overexpression and knockdown of ZFP36 for subsequent rescue experiments (n = 3). In vitro experimental groups: OE-EGR1+si-NC, OE-EGR1+si-ZFP36, OE-EGR1+si-ZFP36+OE-ZFP36. (b–c) Expression levels of glycosphingolipid metabolism markers (HEXA, HEXB, GLA) in macrophages were determined by qRT-PCR and WB (n = 3). Overexpression of EGR1 combined with ZFP36 knockdown downregulates the expression of these markers, while subsequent overexpression of ZFP36 reverses this trend. ( d –e) The proportion of M1 macrophages (CD86 + ) and M2 macrophages (CD163 + ) was determined by flow cytometry, with representative gating plots and quantitative bar charts shown, demonstrating that ZFP36 mediates EGR1 regulation of M1/M2 balance (n = 3). Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs OE-NC/si-NC/OE + EGR1+si-NC/OE + EGR1+si-ZFP36.

Article Snippet: Lentiviral vectors for EGR1 overexpression, EGR1 knockdown (sh-EGR1), and corresponding controls were constructed by GeneChem (Shanghai, China).

Techniques: Quantitative RT-PCR, Over Expression, Knockdown, In Vitro, Expressing, Flow Cytometry

Journal: Neurotherapeutics

Article Title: The EGR1/ZFP36 axis governs glycosphingolipid metabolic reprogramming in monocyte-derived macrophages in guillain-barré syndrome

doi: 10.1016/j.neurot.2026.e00895

Figure Lengend Snippet: Validation of EGR1 targeting ZFP36 to regulate myelination in EAN rat models. (a–b) Knockdown efficiency of ZFP36 as verified by qRT-PCR and WB, confirming successful ZFP36 knockdown in rat sciatic nerve following intrathecal siRNA injection (n = 3). (c) Assessment of hindlimb motor function using a grip strength test, showing that ZFP36 knockdown abrogated EGR1 overexpression-induced improvement in motor function (n = 6). (d) TEM was used to examine the pathological changes in myelin, revealing that ZFP36 knockdown reversed the protective effect of EGR1 on myelin structure. (e–f) qRT-PCR and WB were employed to assess the expression levels of myelin-related markers (MBP, S100B, and MPZ), demonstrating that ZFP36 mediates EGR1 regulation of myelin integrity markers (n = 3). Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs si-NC/EAN + OE-NC/EAN + OE-EGR1.

Article Snippet: Lentiviral vectors for EGR1 overexpression, EGR1 knockdown (sh-EGR1), and corresponding controls were constructed by GeneChem (Shanghai, China).

Techniques: Biomarker Discovery, Knockdown, Quantitative RT-PCR, Injection, Over Expression, Expressing

Journal: Cell Death & Disease

Article Title: IGFBP5 promotes diabetic kidney disease progression by enhancing PFKFB3-mediated endothelial glycolysis

doi: 10.1038/s41419-022-04803-y

Figure Lengend Snippet: A RT–PCR analysis of IGFBP5 mRNA levels in NG- or HG-challenged HUVECs. n = 3. B IGFBP5 mRNA was determined by RT–PCR in the kidney tissues of db/db and db/m mice. n = 6. C , D Western blot analysis and densitometric quantification of IGFBP5 protein levels in the kidney lysates of db/m and db/db mice. n = 6. E RNA-sequencing analysis of IGFBP5 expression in HUVECs isolated from gestational diabetic mothers and non-diabetic mothers. n = 3. F A stable IGFBP5 overexpression (IGFBP5-OE) HUVEC line was established using lentiviral infection and an IGFBP5 overexpression sequence. Autofluorescence (red) showed successful transfection. Scale bar, 100 μm. G Expression of IGFBP5 mRNA in IGFBP5-OE HUVECs compared with the control. n = 3. H RT–PCR analysis of the mRNA levels of ICAM-1, TNF-α, IL-6, and MCP-1 in IGFBP5-OE and control cells under NG conditions. n = 3. I RT–PCR analysis of the mRNA levels of ICAM-1, TNF-α, IL-6, and MCP-1 in siIGFBP5-treated HUVECs under HG conditions. n = 3. J Migration of THP-1 cells cocultured in Transwell systems with IGFBP5-OE or control HUVECs under NG conditions. Scale bar, 100 μm. K Statistical analysis was performed by counting the number of migrated cells in different groups. n = 3. The data are shown as the mean ± SD in all statistical graphs. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, no significance.

Article Snippet: The EGR1 overexpression plasmid and pGL3WT-PFK or pGL3-MT-PFK were transfected into HUVECs with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, RNA Sequencing Assay, Expressing, Isolation, Over Expression, Infection, Sequencing, Transfection, Migration

Journal: Cell Death & Disease

Article Title: IGFBP5 promotes diabetic kidney disease progression by enhancing PFKFB3-mediated endothelial glycolysis

doi: 10.1038/s41419-022-04803-y

Figure Lengend Snippet: RNA-seq analysis was performed on IGFBP5-OE- and HG-stimulated HUVECs. A KEGG pathway analysis of upregulated signaling pathways in IGFBP5-OE cells. B KEGG pathway analysis of upregulated signaling pathways in HG-stimulated HUVECs. C , D RT–PCR analysis of the mRNA levels of PFKFB3 in HG-treated HUVECs and the kidneys of db/db or db/m mice. n = 3. E RNA-sequencing data of EGR1 expression in IGFBP5-OE cells. n = 3. F The consensus EGR1-binding motif identified by WebLogo in the EGR1 ChIP-Seq data. G The target region of the PFKFB3 promoter to which EGR1 binds. H RT–PCR analysis of EGR1 and PFKFB3 mRNA levels in IGFBP5-OE cells. n = 3. I , J Western blot analysis and densitometric quantification of EGR1 and PFKFB3 protein levels in IGFBP5-OE cells under NG conditions. n = 3. K RT–PCR analysis of EGR1 and PFKFB3 mRNA levels in HUVECs transfected with siIGFBP5 under HG conditions. L ChIP-PCR results for PFKFB3. n = 3. M Dual-luciferase reporter assay results. n = 3. The data are shown as the mean ± SD. *** P < 0.001; ns, no significance.

Article Snippet: The EGR1 overexpression plasmid and pGL3WT-PFK or pGL3-MT-PFK were transfected into HUVECs with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

Techniques: RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Binding Assay, ChIP-sequencing, Western Blot, Transfection, Luciferase, Reporter Assay

Journal: Science Advances

Article Title: Egr1 is a 3D matrix–specific mediator of mechanosensitive stem cell lineage commitment

doi: 10.1126/sciadv.abm4646

Figure Lengend Snippet: ( A ) Egr1 mRNA expression kinetics during differentiation within bare (RGD − ) hydrogels and RGD-ligated (RGD + ) 2D and 3D hydrogels. Each level is relative to the expression level on 2D soft gel right after encapsulation (30 min). Hydrogels of 0.1 and 1.2 kPa were used for the soft and stiff conditions, respectively. Expression of mRNA level for Egr1 in the bare soft and stiff hydrogels with RGD sequence–containing peptides or RAD-containing peptides (control) ( B ) and with DMSO (control) or Exo-1 ( C ). ( D ) Western blot and quantification of EGR1 protein expression in NSCs encapsulated within the four different hydrogels (RGD − /soft, RGD − /stiff, RGD + /soft, and RGD + /stiff) for 24 hours. ( E ) Schematic illustration summarizing the stiffness and RGD dependence of Egr1 transcription in 3D gels, with stiffness-dependent Egr1 expression observed only in 3D matrices and independently of RGD-integrin binding. One-way ANOVA followed by Tukey test **** P < 0.001, *** P < 0.005, ** P < 0.01, * P < 0.05. Graphs show means ± SD.

Article Snippet: Retroviral vectors were packaged in HEK 293 T cells with Gag/pol and VSV-G through PEI transfection with plasmids for Egr1 overexpression (pMXs-hs-EGR1, plasmid no. 52724, Addgene) and control (pMXs-puro GFP-p62, plasmid no. 38277, Addgene).

Techniques: Expressing, Encapsulation, Sequencing, Control, Western Blot, Binding Assay

Journal: Science Advances

Article Title: Egr1 is a 3D matrix–specific mediator of mechanosensitive stem cell lineage commitment

doi: 10.1126/sciadv.abm4646

Figure Lengend Snippet: ( A ) Experimental timeline for probing β-catenin signaling during lineage commitment. ( B ) EGR1 expression after shRNA KD. n = 2. Representative immunofluorescence images of naïve, shCtrl, and Egr1 KD cell lines stained for β-tubulin III (green), GFAP (red), and DAPI (blue) along with quantification of neurogenesis in 3D ( C and D ) and 2D ( E and F ) soft (0.1 kPa) and stiff (1.2 kPa) hydrogels. Scale bar, 100 μm. N = 3 to 5. ( G ) Egr1 mRNA levels in shCtrl and shEGR1-1 cell lines in 2D and 3D gels. ( H ) Representative images (left) of immunofluorescence staining for β-catenin (gray), F-actin (green), and DAPI (blue), with corresponding quantification (right) of β-catenin nuclear localization for NSCs encapsulated with RGD − /soft, RGD − /stiff, RGD + /soft, and RGD + /stiff gels. Scale bar, 50 μm. n > 41 cells per group. ( I ) Luciferase assay for β-catenin activity in wild-type (WT) and NSC reporter cells embedded in the four different gels. n = 15 technical replicates including n = 3 biological replicates per each condition. ( J ) Western blotting for active β-catenin (phosphorylated at Ser 552 ) of cells embedded in the four different gels. ( K ) Luciferase assay showing β-catenin activity of shCtrl and shEGR1-1 cells in 3D gels. Values are normalized to shCtrl levels in RGD − gels within each stiffness. ( L ) mRNA expression level by qPCR of Egr1 and three different genes ( Axin1 , Prkaca , and Dvl1 ) involved in Wnt signaling in 2D and 3D gels. The level for each gene is relative to that of shCtrl on 2D soft gels. n = 3 biological replicates. ( M ) Western blotting of active β-catenin (Ser 552 ) of shCtrl and shEGR1-1 cells encapsulated in the four different gels. ( N ) Schematic of the proposed mechanism through which EGR1 acts through β-catenin signaling to regulate stiffness-dependent lineage commitment in 3D. **** P < 0.001, *** P < 0.005, ** P < 0.01, * P < 0.05.

Article Snippet: Retroviral vectors were packaged in HEK 293 T cells with Gag/pol and VSV-G through PEI transfection with plasmids for Egr1 overexpression (pMXs-hs-EGR1, plasmid no. 52724, Addgene) and control (pMXs-puro GFP-p62, plasmid no. 38277, Addgene).

Techniques: Expressing, shRNA, Immunofluorescence, Staining, Luciferase, Activity Assay, Western Blot

Journal: Science Advances

Article Title: Egr1 is a 3D matrix–specific mediator of mechanosensitive stem cell lineage commitment

doi: 10.1126/sciadv.abm4646

Figure Lengend Snippet: ( A ) Representative images of rhodamine-phalloidin–stained NSCs differentiated in RGD-functionalized 2D and 3D gels for 4 hours as a function of matrix stiffness (0.1, 1.2, and >2 kPa). Images for 3D gels were obtained after sectioning. Scale bar, 50 μm. ( B ) Quantification of peak cortex actin intensity line scan after background subtraction (left) and representative images of phalloidin-stained cells in the four different 3D gels and color-coded representation of linearized and zoomed-in view of the cortex for each image (right). Images for 3D gels were obtained after sectioning. Scale bar, 10 μm. ( C ) mRNA expression level of Egr1 after 5 hours of encapsulation with RGD − (left) and RGD + (right) gels after treatment of blebbistatin (1 μM) and cytochalasin D (cyt D, 1 μM) ( n = 3). DMSO was treated as control. ( D ) Quantification of peak cortex actin intensity line scan (left) and images of phalloidin-stained cells in 3D stiff gels under DMSO- (control) and cyt D–treated conditions (right). ( E ) Representative images of immunostaining for β-tubulin III (green), GFAP (red), and DAPI (blue) and quantification of β-tubulin III– and GFAP-positive cells in RGD + gels after treatment with DMSO (control) and cyt D. Scale bar, 50 μm. ( F ) Schematics showing proposed role of actin assembly in regulation of stiffness-dependent Egr1 expression in 3D matrices. One-way ANOVA followed by Tukey test **** P < 0.001, *** P < 0.005, * P < 0.05. Graphs show means ± SD. a.u., arbitrary unit.

Article Snippet: Retroviral vectors were packaged in HEK 293 T cells with Gag/pol and VSV-G through PEI transfection with plasmids for Egr1 overexpression (pMXs-hs-EGR1, plasmid no. 52724, Addgene) and control (pMXs-puro GFP-p62, plasmid no. 38277, Addgene).

Techniques: Staining, Expressing, Encapsulation, Control, Immunostaining

Journal: Science Advances

Article Title: Egr1 is a 3D matrix–specific mediator of mechanosensitive stem cell lineage commitment

doi: 10.1126/sciadv.abm4646

Figure Lengend Snippet: ( A ) Schematics illustrating potential role of confining stress as a 3D-specific regulator of Egr1 expression. ( B ) Representative 3D rendering of NSCs after 3 or 24 hours of encapsulation (top) and cell volumes (bottom). Cells were embedded in RGD − /soft, RGD − /stiff, RGD + /soft, and RGD + /stiff gels; 10 to 77 cells per group. Scale bar, 30 μm. ( C ) ABAQUS simulation to calculate matrix confining stress during cell volumetric growth in the four gel conditions, showing: model system (left), color-coded stress field with the direction of matrix to the cells (center), and quantified stress values with incubation time (right). Close similarities between RGD + and RGD − conditions reflect the RGD independence of measured cell volume, an input parameter. White dotted line represents the cell boundary. ( D ) Schematics depicting application of osmotic pressure to the cells in 3D gels to release confining stress during volumetric growth. Dotted line represents the cell size right after encapsulation. ( E ) Shear elastic moduli of RGD + /soft and RGD + /stiff gels incubated under nontreated (Ctrl) and PEG (1.5 wt %)–treated conditions for 3 hours. ( F ) Cell volume in soft gels with and without PEG (1.5 wt %) and stiff gels without PEG (3 hours). ( G ), Egr1 mRNA expression level changes in RGD + soft and stiff gels under Ctrl and PEG (1.5 wt %) conditions (30 min, 3 hours, and 9 hours). ( H ) Egr1 expression as a function of PEG concentration for cells in suspension and in gels. ( I ) Scatter plot of peak cortex actin intensity versus projected cell area of the cells encapsulated in 3D RGD + /soft (left) and RGD + /stiff (right) gels under Ctrl or PEG (1.5 wt %) condition for 3 hours. ( J ) Representative images of phalloidin-stained cells in sectioned RGD + gels under the Ctrl and PEG conditions and color-coded linearized view of the cortex for each image. Scale bar, 10 μm. **** P < 0.001, *** P < 0.005, ** P < 0.01, * P < 0.05.

Article Snippet: Retroviral vectors were packaged in HEK 293 T cells with Gag/pol and VSV-G through PEI transfection with plasmids for Egr1 overexpression (pMXs-hs-EGR1, plasmid no. 52724, Addgene) and control (pMXs-puro GFP-p62, plasmid no. 38277, Addgene).

Techniques: Expressing, Encapsulation, Incubation, Shear, Concentration Assay, Suspension, Staining

Journal: Science Advances

Article Title: Egr1 is a 3D matrix–specific mediator of mechanosensitive stem cell lineage commitment

doi: 10.1126/sciadv.abm4646

Figure Lengend Snippet: ( A ) Projected area of cell nuclei in the four different 3D gels (RGD − /soft, RGD − /stiff, RGD + /soft, and RGD + /stiff) (left) and corresponding representative images of DAPI-stained cells in the gels after sectioning. N > 69 cells were used for each condition. Scale bar, 10 μm. ( B ) Stiffness dependence of H3K9me3 level in 2D and 3D gels ( n = 3). ( C ) mRNA expression level of Egr1 in the four different 3D gels after treatment of DMSO (control) and JIB-04 (3 μM) for 3 hours. All values are normalized to values for DMSO-treated RGD − /soft gels ( n = 3). ( D ) H3K9me3 level of cells in RGD − and RGD + stiff gels after treatment of DMSO (control) and cyt D (1 μM). ( E ) Proposed mechanism for regulation of Egr1 expression by 3D gel stiffness–dependent confining stress via H3K9 trimethylation. One-way ANOVA followed by Tukey test. **** P < 0.001, *** P < 0.005, ** P < 0.01. Graphs show means ± SD.

Article Snippet: Retroviral vectors were packaged in HEK 293 T cells with Gag/pol and VSV-G through PEI transfection with plasmids for Egr1 overexpression (pMXs-hs-EGR1, plasmid no. 52724, Addgene) and control (pMXs-puro GFP-p62, plasmid no. 38277, Addgene).

Techniques: Staining, Expressing, Control

Journal: Cancers

Article Title: EGR1/GADD45α Activation by ROS of Non-Thermal Plasma Mediates Cell Death in Thyroid Carcinoma

doi: 10.3390/cancers13020351

Figure Lengend Snippet: Transcriptome analysis of THCA cells after NTPAM treatment identifies EGR1 as a regulator of thyroid tumorigenesis. ( A ) Correlation matrix of BCPAP and 8505C cells with or without NTPAM treatment. ( B ) Multi-dimensional scaling plots for BCPAP and 8505C cells with or without NTPAM treatment. ( C ) Heatmap analysis of significantly changed genes related to the UPRmt in BCPAP and 8505C cells with p < 0.05 for comparisons of cells with or without NTPAM treatment. ( D ) The total number of significantly enriched KEGG gene sets with corrected p -values < 0.05 according to the Benjamini-Hochberg procedure (considered statistically significant). ( E ) Top 10 upregulated pathways and top 10 downregulated pathways in BCPAP cells in relation to NTPAM treatment. ( F ) Upstream regulators in relation to NTPAM treatment in BCPAP and 8505C cells via IPA. ( G ) Heatmap analysis of significantly changed genes related to EGR1 with p < 0.05 compared to BCPAP cells with or without NTPAM treatment. ( H ) Box plot of log2TPM of EGR1 values between normal and tumor samples in the TCHA cohort. The significance for between group differences was evaluated using logrnak test. ( I ) Disease-free survival in relation to EGR1 expression in the THCA cohort. TPM: transcripts per million.

Article Snippet: The overexpression vectors for EGR1 and GADD45α were obtained from OriGene Technologies (Rockville, MD, USA).

Techniques: Expressing

Journal: Cancers

Article Title: EGR1/GADD45α Activation by ROS of Non-Thermal Plasma Mediates Cell Death in Thyroid Carcinoma

doi: 10.3390/cancers13020351

Figure Lengend Snippet: NTPAM-induced EGR1 inhibits THCA cell viability. ( A ) Changes in EGR1 expression after NTPAM treatment, as determined by RNA-seq. Results were analyzed using independent t -test. After 2 h dose-NPTAM treatment for 24 h, the EGR1 expression was examined by quantitative PCR (qPCR) analysis ( B ) and western blot analysis ( C ). Results were analyzed using Mann-Whitney U test. ( D ) Tissue samples obtained from patients with THCA were examined using western blot analysis with an anti-EGR1 antibody. ( E ) BCPAP and 8505C cells were transiently transfected with an EGR1 overexpression vector or a negative control vector for 48 h. Cell viability was analyzed by cell-counting analysis. Results were analyzed using Mann-Whitney U test. ( F ) BCPAP and 8505C cells were transiently transfected with EGR1-specific siRNA or negative control siRNA for 48 h. Cell viability was analyzed by cell-counting analysis. Results were analyzed using Mann-Whitney U test. Each figure is representative of three independent experiments. Data were expressed as mean ± standard deviation of the mean (±SD). Differences were considered relevant at p < 0.05 (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The overexpression vectors for EGR1 and GADD45α were obtained from OriGene Technologies (Rockville, MD, USA).

Techniques: Expressing, RNA Sequencing, Real-time Polymerase Chain Reaction, Western Blot, MANN-WHITNEY, Transfection, Over Expression, Plasmid Preparation, Negative Control, Cell Counting, Standard Deviation

Journal: Cancers

Article Title: EGR1/GADD45α Activation by ROS of Non-Thermal Plasma Mediates Cell Death in Thyroid Carcinoma

doi: 10.3390/cancers13020351

Figure Lengend Snippet: NTPAM induces cell death through the regulation of ROS-induced EGR1 in THCA cells. BCPAP and 8505C cells were treated with 2 h dose-NTPAM for 6 h or cotreated with NTPAM and NAC. ( A ) Mitochondrial ROS levels were measured by Mitosox-based flow cytometry. Results were analyzed using non-parametric one-way ANOVA followed by Dunnett’s post-hoc test. ( B ) Cell viability was evaluated using the WST1 assay. Results were analyzed using non-parametric one-way ANOVA followed by Dunnett’s post-hoc test. ( C ) Cell lysates were assessed by western blot analysis using antibodies against EGR1, PARP, and β-actin. ( D ) BCPAP and 8505C cells were treated with NTPAM for 24 h in a dose-dependent manner up to 1:4 dilution and cell lysates were assessed by western blot analysis using an antibody against EGR1. ( E ) BCPAP and 8505C cells were transfected with or without EGR1 vector for 48 h, and then cells were treated with NTPAM for 24 h at the indicated concentrations. Cell viability was analyzed using the WST1 assay. Results were analyzed using Mann-Whitney U test. ( F ) BCPAP and 8505C cells were transfected with or without EGR1-specific siRNA for 48 h, and then cells were treated with NTPAM for 24 h at the indicated concentrations. Cell viability was analyzed using the WST1 assay. Results were analyzed using Mann-Whitney U test. Each figure is representative of three independent experiments. Data were expressed as mean ± standard deviation of the mean (±SD). Differences were considered relevant at p < 0.05 (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The overexpression vectors for EGR1 and GADD45α were obtained from OriGene Technologies (Rockville, MD, USA).

Techniques: Flow Cytometry, Western Blot, Transfection, Plasmid Preparation, MANN-WHITNEY, Standard Deviation

Journal: Cancers

Article Title: EGR1/GADD45α Activation by ROS of Non-Thermal Plasma Mediates Cell Death in Thyroid Carcinoma

doi: 10.3390/cancers13020351

Figure Lengend Snippet: NTPAM induces cell death through the ROS/EGR1/GADD45α pathway in THCA cells. ( A ) Changes in the expression of EGR1 target genes after NTPAM treatment, as determined by RNA-seq. Results were analyzed using independent t -test. After treatment of BCPAP and 8505C cells with 2 h dose-NTPAM for 24 h, GADD45α was examined using qPCR analysis ( B ) and western blot analysis ( C ). Results were analyzed using Mann-Whitney U test. Cells were transfected with EGR1 siRNA ( D ) or the EGR1 overexpression vector ( E ) for 48 h, followed by western blot analysis. EGR1 siRNA ( F ) or the EGR1 overexpression vector ( G ) was co-transfected with the wild-type GADD45α promoter or the mutant GADD45α promoter P-2/3m into 8505C cells. At 24 h after transfection, cells were lysed, and luciferase activity was measured using a luminometer (FilterMax F5). Results were analyzed non-parametric one-way ANOVA followed by Dunnett’s post-hoc test. 8505C cells were transiently transfected with a GADD45α overexpression vector ( H ) or the GADD45α-specific siRNA ( I ) for 48 h. Cell viability was evaluated using the WST1 assay. Results were analyzed using Mann-Whitney U test. Each figure is representative of three independent experiments. Data were expressed as mean ± standard deviation of the mean (±SD). Differences were considered relevant at p < 0.05 (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The overexpression vectors for EGR1 and GADD45α were obtained from OriGene Technologies (Rockville, MD, USA).

Techniques: Expressing, RNA Sequencing, Western Blot, MANN-WHITNEY, Transfection, Over Expression, Plasmid Preparation, Mutagenesis, Luciferase, Activity Assay, Standard Deviation

Journal: Cancers

Article Title: EGR1/GADD45α Activation by ROS of Non-Thermal Plasma Mediates Cell Death in Thyroid Carcinoma

doi: 10.3390/cancers13020351

Figure Lengend Snippet: NTPAM exerts anticancer effects in a xenograft in vivo model. Luciferase-expressing FRO (FRO-Luc) THCA cells were injected subcutaneously into BALB/c-nude mice and treated with NTPAM every day for 3 weeks. ( A ) Schematic illustration of the in vivo experimental design. ( B ) Final tumor images of cancer cells tracked with the IVIS imaging system following the injection of mice with FRO-Luc cells. ( C ) The luminescence radiance of the ROI of the tumor was determined. Results were analyzed using Mann-Whitney U test. ( D ) Final tumor images. ( E ) Final tumor weight. Tumor tissues were extracted after the final determination of tumor growth. Results were analyzed using Mann-Whitney U test. ( F ) Representative images of H&E and immunohistochemical staining of control and NTPAM group. ( G ) The proposed NTPAM-induced ROS/EGR1/GADD45α axis in THCA based on this study. Data were expressed as mean ± standard deviation of the mean (±SD). Differences were considered relevant at p < 0.05 (* p < 0.05).

Article Snippet: The overexpression vectors for EGR1 and GADD45α were obtained from OriGene Technologies (Rockville, MD, USA).

Techniques: In Vivo, Luciferase, Expressing, Injection, Imaging, MANN-WHITNEY, Immunohistochemical staining, Staining, Control, Standard Deviation