egr1 overexpression plasmid  (Thermo Fisher)

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