Journal: Journal of Inflammation Research

Article Title: ETS1 Expression in Diabetic Foot Ulcers: Implications for Fibroblast Phenotype and Wound Healing Through the PP2A/YAP Pathway

doi: 10.2147/JIR.S477470

Figure Lengend Snippet: Effects of ETS1 Downregulation on Migration and Collagen Proliferation in DMFBs. ( A and B ) Detecting the silencing efficiency of fibroblasts from healthy controls by qRT-PCR and Western blotting after siRNA-mediated ETS1 knockdown. ( C and D ) Fibroblasts from ulcer margin of DFUs or healthy controls were cultured, and scratch assays were used to demonstrate the migratory ability after siRNA-mediated ETS1 knockdown. ImageJ was used to analyze the percentage of wound closure. ( E and F ) The relative expressionof ETS1 in fibroblasts as determined by qRT-PCR and Western blotting after siRNA-mediated ETS1 knockdown. TGFβ1 (10ng/ml) intervention was used to induce α-SMA . ImgeJ software was used for statistical analysis. Data are presented as mean ± SD values of three independent experiments. *, P < 0.05.

Article Snippet: Small interfering RNA (siRNA) duplexes specific to ETS1 (Cat#: sc-29309) and a non-targeting scrambled control (Cat#: sc-37007) were procured from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Migration, Quantitative RT-PCR, Western Blot, Knockdown, Cell Culture, Software

Journal: Journal of Inflammation Research

Article Title: ETS1 Expression in Diabetic Foot Ulcers: Implications for Fibroblast Phenotype and Wound Healing Through the PP2A/YAP Pathway

doi: 10.2147/JIR.S477470

Figure Lengend Snippet: ETS1 Regulation of YAP through Targeting PP2A. ( A ) Utilizing the UCSC and JASPAR databases to predict the binding sites between human ETS1 and the PP2A promoter, The binding intensity scores, binding segment start and end points, as well as gene sequences are illustrated. ( B ) The relative expressionof ETS1 and PP2A in fibroblasts from DFU Wagner II–IV determined by qRT-PCR. ( C ) Comparison the expression change between ETS1 and PP2A after siRNA-mediated ETS1 knockdown. ( D ) Detecting the ETS1 expression of fibroblasts from healthy controls by Western blotting after siRNA-mediated ETS1 knockdown. ( E ) Fibroblasts from DFU ulcer margins or healthy controls were treated with siETS1, and the expression of PP2A-B and phosphorylated YAP (p-YAP) was analyzed by Western blotting. The siETS1 means siRNA-mediated ETS1 knockdown, whereas the siRNA means a non-targeting scrambled siRNA as control. Data are presented as mean ± SD values of three independent experiments. *, P < 0.05.

Article Snippet: Small interfering RNA (siRNA) duplexes specific to ETS1 (Cat#: sc-29309) and a non-targeting scrambled control (Cat#: sc-37007) were procured from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Binding Assay, Quantitative RT-PCR, Comparison, Expressing, Knockdown, Western Blot, Control

Journal: Journal of Inflammation Research

Article Title: ETS1 Expression in Diabetic Foot Ulcers: Implications for Fibroblast Phenotype and Wound Healing Through the PP2A/YAP Pathway

doi: 10.2147/JIR.S477470

Figure Lengend Snippet: The Expression of ETS1 in DFUs . ( A ) For DFUs of Wagner II–IV, HE staining was used to analyze their histopathological characteristics, and Masson staining was applied to examine collagen expression (n=6). ( B ) ImageJ software was used to quantify collagen in different DFUs of Wagner II–IV. ( C and D ) Expression of ETS1 mRNA or protein in DFUs Wagner II–IV by qRT-PCR or Western blotting(n=3). ( E ) The localization and expression of ETS1 in DFUs Wagner II–IV as determined by immunofluorescence (n=3). Data are presented as mean ± SD. *, P < 0.05.

Article Snippet: Small interfering RNA (siRNA) duplexes specific to ETS1 (Cat#: sc-29309) and a non-targeting scrambled control (Cat#: sc-37007) were procured from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Staining, Software, Quantitative RT-PCR, Western Blot, Immunofluorescence

Journal: Journal of Inflammation Research

Article Title: ETS1 Expression in Diabetic Foot Ulcers: Implications for Fibroblast Phenotype and Wound Healing Through the PP2A/YAP Pathway

doi: 10.2147/JIR.S477470

Figure Lengend Snippet: The expression of ETS1 from ulcer margin of DFUsor healthy controls. ( A and B ) The relative expression of Ets1 and α-SMA in fibroblasts determined by immunofluorescence. TGFβ1 (10ng/ml) intervention was used to induce α-SMA expression. ImgeJ software was used for statistical analysis. ( C ) The relative expression of α-SMA, ETS1 and CollagenI in fibroblasts normalized to ACTB determined by qRT-PCR. ( D ) Ets1, α-SMA, and Collagen I expression in fibroblasts from Wagner II–IV DFUs were analyzed by Western blotting, and ImageJ software was used for statistical analysis.Data are presented as mean ± SD values of three independent experiments. *, P < 0.05.

Article Snippet: Small interfering RNA (siRNA) duplexes specific to ETS1 (Cat#: sc-29309) and a non-targeting scrambled control (Cat#: sc-37007) were procured from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Immunofluorescence, Software, Quantitative RT-PCR, Western Blot

Journal: Journal of Inflammation Research

Article Title: ETS1 Expression in Diabetic Foot Ulcers: Implications for Fibroblast Phenotype and Wound Healing Through the PP2A/YAP Pathway

doi: 10.2147/JIR.S477470

Figure Lengend Snippet: Effects of ETS1 Downregulation on Migration and Collagen Proliferation in DMFBs. ( A and B ) Detecting the silencing efficiency of fibroblasts from healthy controls by qRT-PCR and Western blotting after siRNA-mediated ETS1 knockdown. ( C and D ) Fibroblasts from ulcer margin of DFUs or healthy controls were cultured, and scratch assays were used to demonstrate the migratory ability after siRNA-mediated ETS1 knockdown. ImageJ was used to analyze the percentage of wound closure. ( E and F ) The relative expressionof ETS1 in fibroblasts as determined by qRT-PCR and Western blotting after siRNA-mediated ETS1 knockdown. TGFβ1 (10ng/ml) intervention was used to induce α-SMA . ImgeJ software was used for statistical analysis. Data are presented as mean ± SD values of three independent experiments. *, P < 0.05.

Article Snippet: Small interfering RNA (siRNA) duplexes specific to ETS1 (Cat#: sc-29309) and a non-targeting scrambled control (Cat#: sc-37007) were procured from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Migration, Quantitative RT-PCR, Western Blot, Knockdown, Cell Culture, Software

Journal: Journal of Inflammation Research

Article Title: ETS1 Expression in Diabetic Foot Ulcers: Implications for Fibroblast Phenotype and Wound Healing Through the PP2A/YAP Pathway

doi: 10.2147/JIR.S477470

Figure Lengend Snippet: ETS1 Regulation of YAP through Targeting PP2A. ( A ) Utilizing the UCSC and JASPAR databases to predict the binding sites between human ETS1 and the PP2A promoter, The binding intensity scores, binding segment start and end points, as well as gene sequences are illustrated. ( B ) The relative expressionof ETS1 and PP2A in fibroblasts from DFU Wagner II–IV determined by qRT-PCR. ( C ) Comparison the expression change between ETS1 and PP2A after siRNA-mediated ETS1 knockdown. ( D ) Detecting the ETS1 expression of fibroblasts from healthy controls by Western blotting after siRNA-mediated ETS1 knockdown. ( E ) Fibroblasts from DFU ulcer margins or healthy controls were treated with siETS1, and the expression of PP2A-B and phosphorylated YAP (p-YAP) was analyzed by Western blotting. The siETS1 means siRNA-mediated ETS1 knockdown, whereas the siRNA means a non-targeting scrambled siRNA as control. Data are presented as mean ± SD values of three independent experiments. *, P < 0.05.

Article Snippet: Small interfering RNA (siRNA) duplexes specific to ETS1 (Cat#: sc-29309) and a non-targeting scrambled control (Cat#: sc-37007) were procured from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Binding Assay, Quantitative RT-PCR, Comparison, Expressing, Knockdown, Western Blot, Control

Journal: bioRxiv

Article Title: B lymphocytes acquire myeloid and autoimmune phenotypes via the downregulation of lymphocyte-specific protein-1

doi: 10.1101/2024.06.28.600734

Figure Lengend Snippet: a, Heatmap displaying the DEGs between WT and Lsp1 -/- B cells stimulated with αIgM or LPS for 4 hours. Red and blue indicate up- and downregulation, respectively (left panel). The right panel shows the hematopoietic cell markers upregulated by Lsp1 deficiency. b , Upregulation of myeloid gene expression in Lsp1 -/- B cells, as determined by qRT‒PCR; the fold changes were calculated using the 2 -ΔΔCt method. c, Percentage of MPO + cells in splenic B cells freshly isolated from WT and Lsp1 -/- mice (n=20 per group), as determined by immunofluorescence staining with anti-B220 (green) and anti-MPO (red) Abs. d - i, Splenic B cells isolated from WT and Lsp1 -/- mice were stimulated with αIgM or LPS for 24 hours ( d-f ), 7 days ( g ), or 5 days ( h,i ). d , Immunoblot (left panel) and ELISA (right panel, n=10 per group) analyses of MPO expression. e , Intracellular ROS production (n=7 per group) by flow cytometry using H 2 DCFDA. f , Production of IL-1β and TNF-α (n=9∼10) by ELISA. g , Percentage of CD11c + cells in WT and Lsp1 -/- B cells (n=9∼10) by flow cytometry. A representative zebra plot for CD11c + cells (red dotted box) is presented on the left. h , Immunoblotting for Blimp1 and Xbp1, which are representative TFs involved in B-cell differentiation. i , ELISA for IgG, IgG2c, and IgG3 produced by B cells stimulated with LPS (n=3 per group). j,k, LSP1 -regulated gene expression and IgG production in human B cells. Sorted human B cells were electroporated with LSP1 siRNA ( j ) or an LSP1-EGFP plasmid ( k ). After 24 hours, the mRNA expression levels of LSP1 , MPO , IL1B , and CYBB in the cells were determined via qRT‒PCR. IgG production by B cells stimulated with αIgM, CD40L, and CpG for 5 days was determined via ELISA. l, GSEA plots demonstrating significant enrichment between the Lsp1 -regulated genes in Clusters 1 and 2 and the genes (up- and downregulated) defining CD11b + B cells, CD11c + B cells, or ABCs. NES=normalized enrichment score. The data in the bar graphs are presented as the means ± SDs of at least two independent experiments. P values were determined by multiple unpaired two-tailed t tests ( b to g ), two-way ANOVA with Sidak’s multiple comparisons ( i , right panels in j and k ), and the Mann‒Whitney U or unpaired t test (left panels in j and k ). * P < 0.05; ** P < 0.01; *** P < 0.001; and **** P < 0.0001.

Article Snippet: For gene knockdown, human LSP1 siRNA (Santa Cruz Biotechnology, #sc-42899), ETS1 siRNA (Santa Cruz Biotechnology #sc-29309), or mouse PKCβ siRNA (Santa Cruz Biotechnology, #sc-36255) was electroporated into B cells using an Amaxa Nucleofector (Lonza) and incubated as indicated.

Techniques: Gene Expression, Isolation, Immunofluorescence, Staining, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Cell Differentiation, Produced, Plasmid Preparation, Two Tailed Test

Journal: bioRxiv

Article Title: B lymphocytes acquire myeloid and autoimmune phenotypes via the downregulation of lymphocyte-specific protein-1

doi: 10.1101/2024.06.28.600734

Figure Lengend Snippet: a, Venn diagram displaying transcription factors (TFs) that have a high potential to regulate the 369 upregulated DEGs in Lsp1 -/- B cells with or without αIgM or LPS treatment (left panel). The bar graph on the right depicts the numbers of C/EBP target genes among the DEGs in Lsp1 -/- B cells. b, PPI network representing the interactions between C/EBPα/β and the 31 C/EBP target genes involved hematopoietic cell lineage markers, cytokines/chemokines, and phagocytosis. The color of the nodes indicates the fold change in the genes affected by Lsp1 deficiency. c, d, Quantitative RT‒PCR analysis of Cebpa and Cebpb mRNA expression (n=4∼7; c ) and immunoblotting for C/EBPβ protein expression ( d ). Lsp1 -/- versus WT B cells were stimulated with αIgM or LPS for 4 hours ( c ) or for 24 hours ( d ). e, f, Lsp1 -/- B cells were transduced with control or Cebpb shRNAs, and the cells were then stimulated with αIgM or LPS for 24 hours for C/EBPβ immunoblotting ( e ) and MPO and TNF-α ELISAs (n=4; f ). g, Control or Cebpb shRNA-treated Lsp1 -/- B cells were stimulated with αIgM or LPS for 5 days, after which Blimp1, IRF4, and Xbp1 expression was determined via immunoblot assay. h, Immunoblots of phosphorylated PKCβ (pPKCβ) in Lsp1 -/- versus WT B cells. Splenic B cells were stimulated with αIgM or LPS for 5 minutes. i, j, Effects of PKCβ inhibition on C/EBPβ and IL-1β expression in Lsp1 -/- B cells stimulated with αIgM or LPS. ( i ) Lsp1 -/- B cells were pretreated with the PKCβ inhibitor ruboxistaurin (4 μM) for 1 hour and stimulated with αIgM or LPS for 24 hours. ( j) Lsp1 -/- B cells were stimulated with αIgM or LPS for 24 hours and then electroporated with control or Prkcb siRNA for an additional 24 hours. The expression levels of C/EBPβ, its target gene IL-1β, and PKCβ were determined via an immunoblot assay. k, Proposed model for the dual regulation of B-cell functions by LSP1. The data in the bar graphs are presented as the means ± SDs of at least three independent experiments. P values were determined by multiple unpaired t tests ( c ) and paired t tests ( f ). * P < 0.05; ** P < 0.01.

Article Snippet: For gene knockdown, human LSP1 siRNA (Santa Cruz Biotechnology, #sc-42899), ETS1 siRNA (Santa Cruz Biotechnology #sc-29309), or mouse PKCβ siRNA (Santa Cruz Biotechnology, #sc-36255) was electroporated into B cells using an Amaxa Nucleofector (Lonza) and incubated as indicated.

Techniques: Expressing, Western Blot, Transduction, Control, shRNA, Inhibition

Journal: bioRxiv

Article Title: B lymphocytes acquire myeloid and autoimmune phenotypes via the downregulation of lymphocyte-specific protein-1

doi: 10.1101/2024.06.28.600734

Figure Lengend Snippet: a,b, Effects of pristane on the expression of LSP1 and its target genes. WT mice were intraperitoneally injected with 500 μl of pristane or PBS as a control (n=4 per group). Two weeks later, the expression of LSP1, its upstream regulator ETS1, and its downstream target genes C/EBPβ and MPO in splenic B cells was determined. ( a ) Immunoblot analysis. The numbers indicate the individual mice. ( b ) Flow cytometry. The percentages of B-cell subpopulations, including total CD19 + B cells, GC B cells (GL7 + CD95 + in CD19 + cells), ABCs (CD11b + CD11c + in CD19 + cells), and plasma cells (CD19 - CD138 + cells), were analyzed. A representative pseudocolor plot (left panel) shows the ABC population in the rectangles with red dots in the PBS (n=6) and pristane (n=8) groups. c-h, Aggravation of pristane-induced lupus by Lsp1 deficiency. WT (n=7) and Lsp1 -/- (n=8) mice were intraperitoneally injected with pristane. ( c ) Changes in body weight. ( d ) Diffuse pulmonary hemorrhage (DPH) severity in the lungs and bronchoalveolar lavage fluids (BALFs) of WT (n=12) and Lsp1 -/- (n=17) mice was evaluated via gross inspection. DPH incidence and DPH scores of the two groups were also determined by H&E staining. Infiltration of CD45 + leukocytes ( e ) and IgG expression ( f ) in the lungs of WT and Lsp1 -/- mice were assessed by immunohistochemistry using anti-CD45 and anti-IgG Abs. The numbers of CD45 + cells were manually counted from 5 fields per slide. The intensity of IgG expression in 3 fields per lung tissue sample was analyzed using ImageJ. g , The number of ABCs and plasma cells in the BALF of WT and Lsp1 -/- mice was assessed via flow cytometry. h , ELISA for anti-dsDNA IgG levels in the sera of WT (n=14) and Lsp1 -/- (n=19) mice. i-k, Acceleration of pristane-induced lupus by B-cell-specific depletion of Lsp1 . Lsp1 fl/fl and Lsp1 fl/fl Cd19 cre/- mice were intraperitoneally injected with pristane. ( i ) DPH severity in the lungs and BALF of the two groups of mice (n=19 per group). ( j) Infiltration of CD45 + cells in the lungs of the two groups (n=15 per group). ( k ) Anti-dsDNA IgG production in the sera of Lsp1 fl/fl (n=20) and Lsp1 fl/fl Cd19 cre/- (n=19) mice. The data in the bar graphs are presented as the means ± SDs of at least three independent experiments. P values were determined by the Mann‒Whitney U test ( b, d to k, except for incidence data), two-way ANOVA with Sidak’s multiple comparisons ( c ), and Fisher’s exact test (percentage of incidence in d and i ). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: For gene knockdown, human LSP1 siRNA (Santa Cruz Biotechnology, #sc-42899), ETS1 siRNA (Santa Cruz Biotechnology #sc-29309), or mouse PKCβ siRNA (Santa Cruz Biotechnology, #sc-36255) was electroporated into B cells using an Amaxa Nucleofector (Lonza) and incubated as indicated.

Techniques: Expressing, Injection, Control, Western Blot, Flow Cytometry, Clinical Proteomics, Staining, Immunohistochemistry, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: B lymphocytes acquire myeloid and autoimmune phenotypes via the downregulation of lymphocyte-specific protein-1

doi: 10.1101/2024.06.28.600734

Figure Lengend Snippet: a, Flow cytometry was used to determine LSP1 expression levels in the peripheral B cells of healthy donors (HD, n=20) and SLE patients (n=18): MFI=mean fluorescence intensity. Disease activity was evaluated by the SLEDAI. b, LSP1 expression in cultured B cells from HDs (n=4) versus SLE B cells (n=4∼5) determined by flow cytometry. B cells were stimulated with αIgM, CD40L, and CpG for 3 days. c, qRT‒PCR analysis of ETS1 mRNA expression in B cells from HDs (n=18) and SLE patients (n=17, left panel) and its correlation with LSP1 mRNA expression in HDs (black circle) and SLE patients (blue circle, right panel). d,e, Regulation of LSP1 expression by ETS1. d , ETS1 and LSP1 mRNA expression in human B cells transfected with ETS1 siRNA, as evaluated by qRT‒PCR (n=4). e , qRT‒PCR analysis of Ets1 and Lsp1 mRNA in B cells from WT ( Ets1 fl/fl , n=8) and Ets1 -/- ( Ets1 fl/fl C d19 Cre/- , n=4) mice. f, LSP1 expression in WT B cells (n=6∼7) stimulated with αIgM and LPS for 5 days in the presence or absence of (pretreated) 20 μM hydralazine, 20 μM procainamide, or 1 μM 5-azacytidine, as examined by flow cytometry. g, TEM images of B cells from HDs and SLE patients. h, i, Immunofluorescence staining of MPO in HDs and SLE B cells using anti-MPO (red) and anti-CD19 (green) Abs and DAPI (blue). h , Representative images of MPO + B cells are indicated by white arrows. i , Percentage of MPO + B cells determined by analyzing 6 fields per sample (n=5 for HDs, n=4 for SLE patients). j, qRT‒PCR and ELISA results for MPO. Left: MPO mRNA expression in HDs (n=17) versus SLE B cells (n=15). Right: ELISA for MPO production by B cells from HDs (n=7) and SLE patients (n=5) activated with the indicated stimuli for 3 days. k, Flow cytometry analysis of LSP1 and MPO expression in CD19 + B cells from HDs (black circle, n=4) and SLE patients (blue circle, n=7). l, Left: LSP1 signature in SLE patients and its correlation with the type I IFN signature: n=24 for the low IFN signature, n=75 for the high IFN signature, and n=181 for HD. Bulk RNA-seq data from SLE PBMCs were obtained from a public database (GSE72509). Right: Estimated population size of monocytic cells by cellular deconvolution analysis. Each dot represents an SLE patient or HD. m , LSP1 signature score in B cells from SLE patients versus HDs determined by pseudobulk RNA-seq analysis (GSE174188; s ee Methods ). n, Uniform Manifold Approximation and Projection (UMAP) plot visualizing the B-cell subsets with high LSP1 scores (>0.8) in SLE patients. The data in the bar graphs are presented as the means ± SDs of at least two independent experiments. P values were determined by the Mann‒Whitney U test (left panels in a , c , and j ; all data in i ); one-way ANOVA with Dunnett’s multiple comparisons (right panel in a ); two-way ANOVA with Sidak’s, Dunnett’s multiple comparison or multiple t test ( b , d - f ; right panel in j ); and the Spearman correlation test (right panels in c and k ). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: For gene knockdown, human LSP1 siRNA (Santa Cruz Biotechnology, #sc-42899), ETS1 siRNA (Santa Cruz Biotechnology #sc-29309), or mouse PKCβ siRNA (Santa Cruz Biotechnology, #sc-36255) was electroporated into B cells using an Amaxa Nucleofector (Lonza) and incubated as indicated.

Techniques: Flow Cytometry, Expressing, Fluorescence, Activity Assay, Cell Culture, Transfection, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Comparison

Journal: bioRxiv

Article Title: B lymphocytes acquire myeloid and autoimmune phenotypes via the downregulation of lymphocyte-specific protein-1

doi: 10.1101/2024.06.28.600734

Figure Lengend Snippet: a , Immunoblotting for LSP1 expression in B cells after ATRA treatment. Splenic B cells were treated with 100 or 1000 nM ATRA for 3 days without stimuli (left panel) or stimulated with αIgM and CD40L in the absence or presence of 100 nM ATRA for 3 days (right panel). b. Quantitative RT-PCR analysis of Lsp1 and Ets1 mRNA expression in splenic B cells treated with 1000 nM ATRA for indicated time (n=5∼6). c-f, Suppressive effects of ATRA on pristane-induced lupus. ( c,d ) One hour before pristane injection, 0.5 mg/kg ATRA (n=17) or PBS (n=19) was administered via the intraperitoneal route to WT mice. Two weeks after pristane injection, lung tissues were harvested and subjected to H&E staining and immunostaining. Lung pathology was evaluated by DPH incidence and score ( c ) and by the extent of infiltration of CD45 + leukocytes ( d ), as described in and . ( e ) Immunoblotting for LSP1 and its target C/EBPβ and MPO in splenic B cells from mice with pristane-induced lupus. The numbers indicate the individual mice. ( f ) Percentages of splenic CD19 + B cells and CD11b + CD11c + in CD19 + cells (ABCs) determined by flow cytometry (n=12 for the PBS group, n=11 for the ATRA group). g, ATRA-induced upregulation of LSP1 expression in SLE B cells. After treatment with ATRA for 3 days, LSP1 expression in the B cells was determined by flow cytometry (top) and western blotting (bottom). h-i, Downregulation of MPO expression in SLE B cells induced by ATRA treatment. PBMCs were serially obtained from SLE patients before and 3 to 6 months after ATRA treatment. ( h ) LSP1 and MPO expression in PBMCs was examined by immunoblotting. ( i ) Immunofluorescence staining of MPO. Representative images of MPO expression (red) in CD19 + B cells (green) are depicted in merged images (white arrows). The numbers of MPO + cells among the CD19 + cells were evaluated in 6 fields per sample. Scale bars=5 μm. j, Efficacy of ATRA treatment in 5 SLE patients who were unresponsive to conventional therapy. The data in the bar graphs in ( b - d , f , and i ) are presented as the means ± SDs of at least two independent experiments. P values were determined by Kruskal-Wallis test with Dunn’s multiple comparison ( b ), Fisher’s exact test (percentage of DPH incidence in c ), an unpaired two-tailed t test ( c, d, and f, except for data on incidence), and a paired t test ( i ). n.s. , not significant; * P < 0.05; ** P < 0.01.

Article Snippet: For gene knockdown, human LSP1 siRNA (Santa Cruz Biotechnology, #sc-42899), ETS1 siRNA (Santa Cruz Biotechnology #sc-29309), or mouse PKCβ siRNA (Santa Cruz Biotechnology, #sc-36255) was electroporated into B cells using an Amaxa Nucleofector (Lonza) and incubated as indicated.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Injection, Staining, Immunostaining, Flow Cytometry, Immunofluorescence, Comparison, Two Tailed Test

Journal: iScience

Article Title: Ets1 facilitates EMT/invasion through Drp1-mediated mitochondrial fragmentation in ovarian cancer

doi: 10.1016/j.isci.2023.107537

Figure Lengend Snippet: Ets1 oncoprotein causes fragmentation of mitochondria (A) Confocal microscopy-based mitochondrial imaging using MitoTracker Red (MTR) is performed in control (untransfected) and Ets1-overexpressing OAW42 cells (n =3 ). Short/fragmented mitochondria have been color-coded with GREEN, and long/fused mitochondria have been coded with RED color in the representative images. (B) Mitochondrial length estimation is performed using LASX software and represented as histogram. (C and D) MTR-based mitochondrial length estimation is performed in control, Ets1-overexpressing and Ets1-knockdown SKOV3 cells (n =3 ). Mitochondrial length estimation is performed on LASX software. (E) Mitotracker Green (MTG)-based live-cell imaging is performed for control and Ets1-overexpressing SKOV3 cells. Data expressed in ±SEM, and statistical significance is calculated using two-tailed Student’s t test (B) and one-way ANOVA (followed by Tukey post hoc analysis) (D) ((∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). NS non-significant. Scale bars 20 μm (A, C), 10 μm (E), also see Figure S1 .

Article Snippet: siRNA for Ets1 , Santa Cruz , Sc-29309.

Techniques: Confocal Microscopy, Imaging, Control, Software, Knockdown, Live Cell Imaging, Two Tailed Test

Journal: iScience

Article Title: Ets1 facilitates EMT/invasion through Drp1-mediated mitochondrial fragmentation in ovarian cancer

doi: 10.1016/j.isci.2023.107537

Figure Lengend Snippet: Mitochondrial fission gene DRP1 is augmented through Ets1-mediated transcriptional regulation (A) RT2 Profiler PCR array is performed for Ets1-overexpressing SKOV3 cells compared to control cells. Comparative enrichment of the genes is depicted as Log 2 fold change w.r.t expression in control cells (n =3 ). (B) Quantitative RT-PCR for Ets1, DRP1, MFN1, MFN2 and OPA1 mRNAs in Ets1-overexpressing SKOV3 cells is performed. The changes in gene expression w.r.t 18s ribosomal RNA are represented as Log 2 fold change (n =3 ). (C–F) Western blot analysis for Drp1, MFN1, and MFN2 proteins is performed for Ets1-overexpressing and knockdown (siRNA-mediated) SKOV3 cells. Control represents non-transfected cells, and scrambled represents scrambled siRNA-transfected SKOV3 cells. α-tubulin serves as loading control. Densitometric analysis has been performed on ImageJ software and represented as histogram (n =3 ). (G) Western blot analysis for Ets1 and Drp1 proteins in IOSE, PA1, SKOV3, OAW42 cell lines, respectively. α-tubulin serves as loading control. Densitometric analysis has been performed on ImageJ software. Drp1/Ets1 ratio in IOSE cells v/s PA1, SKOV3 OAW42 ovarian cancer cells has been depicted as histogram (n =4 ). (H) Immunohistochemistry performed in non-malignant v/s high grade serous ovarian carcinoma (HGSOC) tissue sections for Drp1 [Alexa Fluor 488(GREEN)] and Ets1 [Alexa Fluor 568(R)] internal antigens (n =5 ). (I) Pearson’s correlation coefficient is calculated on LASX software and represented as dot plot. (J) Chromatin Immunoprecipitation for Ets1 transcription factor followed by PCR using proximal primer (P2) and distal primer (P4) for Drp1 promoter is performed. Representative gel image for PCR data has been shown. Input represents complete cross-linked DNA (positive control) and IgG represents immunoprecipitated DNA with rabbit IgG (Negative control). (K) Chromatin immunoprecipitation is performed for Ets1 overexpression and silencing (knockdown) in SKOV3 and PA1 cells. Representative gel images have been shown. Data represented as ±SEM, and statistical significance was calculated using two-tailed Student’s t test (B, D-F) and one-way ANOVA (followed by Tukey post hoc analysis) (G) (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). NS non-significant. Scale bars 20μm (H), also see Figure S2 .

Article Snippet: siRNA for Ets1 , Santa Cruz , Sc-29309.

Techniques: Control, Expressing, Quantitative RT-PCR, Gene Expression, Western Blot, Knockdown, Transfection, Software, Immunohistochemistry, Chromatin Immunoprecipitation, Positive Control, Immunoprecipitation, Negative Control, Over Expression, Two Tailed Test

Journal: iScience

Article Title: Ets1 facilitates EMT/invasion through Drp1-mediated mitochondrial fragmentation in ovarian cancer

doi: 10.1016/j.isci.2023.107537

Figure Lengend Snippet: Ets1-induced mitochondrial fragmentation is through Drp1 upregulation and activating phosphorylation (A and C) MTR-based confocal imaging for understanding mitochondrial fragmentation on Drp1 inhibition through siRNA-mediated silencing (A.) and MDiVi-1 treatment (14μm) for 24h (C.) is done for SKOV3 cells (n =3 ). (B) Mitochondrial size estimation on Drp1 silencing in SKOV3 cells is done using LASX software and represented as histogram. (D) Immunocytochemistry based internal antigen analysis for p-DRP ser616 [Alexa 488 (GREEN)] is done in SKOV3 cells. (E) Estimation of Mean Fluorescence Intensity (MFI) for p-DRP ser616 [Alexa 488 (GREEN)] is done through LASX software and represented as dot plots. Image colocalization analysis for mitochondria (MTR) and p-DRP ser616 [Alexa 488 (GREEN)] has been done through LASX software. Percent co-localization and overlap coefficient have been depicted as dot plots. (F) Western blot analysis for p-DRP ser616 , p-DRP ser637 and total Drp1 (Tot Drp1) is performed for Ets1-overexpressing SKOV3 cells. α-tubulin serves as loading control. Densitometric analysis has been performed on ImageJ software and represented as histogram (n =4 ). (G) MTR-based mitochondrial imaging for SKOV3 cells on treatment with U0126 (10 μM) has been done. Data are expressed in ±SEM, and statistical significance was calculated using two-tailed Student’s t test (E, F) and one-way ANOVA (followed by Tukey post hoc analysis) (B) (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). NS stands for non-significant. Scale bars 10 μM (A., C., D., G., J.), 20μM (E.), also see Figure S3 .

Article Snippet: siRNA for Ets1 , Santa Cruz , Sc-29309.

Techniques: Phospho-proteomics, Imaging, Inhibition, Software, Immunocytochemistry, Fluorescence, Western Blot, Control, Two Tailed Test

Journal: iScience

Article Title: Ets1 facilitates EMT/invasion through Drp1-mediated mitochondrial fragmentation in ovarian cancer

doi: 10.1016/j.isci.2023.107537

Figure Lengend Snippet: Drp1 is recruited at [Endoplasmic Reticulum (ER)-Mitochondria contact sites] (ERMCs) to facilitate Ets1-induced mitochondrial fission (A) Confocal microscopy-based live-cell videography using MitoTracker Green [(MTG) GREEN] for mitochondrial staining and ER Blue-White DPX dye (BLUE) for ER staining is done for Ets1 overexpression in OAW42 cells. Bright red arrows indicate sites of mitochondrial fission. (B) Image co-localization analysis (ICA) has been performed using LASX software. Pearson’s correlation, overlap coefficient, and colocalization rate are represented as dot plots. (C) Line-scan analysis for co-localization of ER (BLUE) and mitochondria (GREEN) has been done using ImageJ software. (D) Confocal microscopy-based live cell imaging using MTG (GREEN) and ER Blue-White DPX dye (BLUE) is done for Ets1 overexpressing SKOV3 cells (n =2 ). (E) ICA is done using LASX software and Pearson’s correlation, overlap coefficient and colocalization rate have been represented as dot plots. (F) Immunocytochemistry-based analysis for internal antigen Drp1 (GREEN), mitochondria stained with MTR (RED) and ER stained with ER Blue-White DPX (BLUE) is performed (n =3 ). White arrows indicate sites of Drp1 recruitment on ERMCs. (G) Line-scan analysis for colocalization is done using ImageJ software along the white-line drawn in (F.) Data are expressed in ±SEM, and statistical significance is calculated using two-tailed Student’s t test. NS stands for non-significant. Scale bars 10μM (A., D.), 20μM (F.), also see Figure S4 .

Article Snippet: siRNA for Ets1 , Santa Cruz , Sc-29309.

Techniques: Confocal Microscopy, Staining, Over Expression, Software, Live Cell Imaging, Immunocytochemistry, Two Tailed Test

Journal: iScience

Article Title: Ets1 facilitates EMT/invasion through Drp1-mediated mitochondrial fragmentation in ovarian cancer

doi: 10.1016/j.isci.2023.107537

Figure Lengend Snippet: Ets1-mediated mitochondrial fragmentation results in mitochondrial load reduction in ovarian cancer (A and B) Flow cytometry based mitochondrial load analysis using MitoTracker Green (MTG) has been performed in OAW42 (n =3 ) and SKOV3 (n =5 ). Fold change and direct values for Mean Fluorescence Intensity (MFI) are depicted as histogram. (C and D) Western blot analysis for VDAC1 mitochondrial marker protein is performed on Ets1 overexpression and silencing in OAW42 and SKOV3 cells. Control stands for untransfected cells and Scrambled stands for scramble siRNA transfection. Densitometric quantitation for VDAC1 expression in SKOV3 cells is done using ImageJ software and depicted as histogram. α-tubulin serves as loading control. (E and F) Western blot analysis for OXPHOS complex proteins in response to Ets1 overexpression and siRNA silencing is done (n =4 ). Control stands for untransfected cells. Scrambled stands for scrambled siRNA transfection. α-tubulin serves as loading control. Densitometric analysis for relative protein expression is done using ImageJ software and plotted in the form of histogram. (G) Western blot analysis for cytoplasmic and mitochondrial protein fractions has been done for OXPHOS complex proteins (n = 5). VDAC serves as mitochondrial marker. GAPDH serves as cytoplasmic marker. Relative protein expression in mitochondrial fraction w.r.t. VDAC is calculated through densitometric analysis using ImageJ software and depicted as histogram. (H) Flow cytometry-based MTG analysis for mitochondrial load assessment has been performed in response to MDiVi-1 treatment (14μm) for 24 h. Changes in Mean Fluorescence Intensity is depicted as histogram (n =3 ). Data are expressed in ±SEM, and statistical significance has been calculated using two-tailed Student’s t test (A-B, D, G) and one-way ANOVA (followed by Tukey post hoc analysis) (F, H) ((∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). NS stands for non-significant, also see Figure S5 .

Article Snippet: siRNA for Ets1 , Santa Cruz , Sc-29309.

Techniques: Flow Cytometry, Fluorescence, Western Blot, Marker, Over Expression, Control, Transfection, Quantitation Assay, Expressing, Software, Two Tailed Test

Journal: iScience

Article Title: Ets1 facilitates EMT/invasion through Drp1-mediated mitochondrial fragmentation in ovarian cancer

doi: 10.1016/j.isci.2023.107537

Figure Lengend Snippet: Ets1 induces mitochondrial bioenergetic alterations through Drp1 regulation in ovarian cancer (A) Seahorse extracellular flux analyzer-based Real-Time ATP Rate assay has been performed in Ets1-overexpressing OAW42 (n =5 ) and SKOV3 (n =5 ) cells. ATP production rate is represented as histogram. The proportions of mitoATP and glycoATP have been color-coded as BLUE and RED, respectively. (B) Extracellular flux analyzer-based Mito Stress test is performed in OAW42 cells in response to Ets1 overexpression and silencing. (C and D) Estimated changes in basal and maximal respiration are represented as histogram. Scrambled stands for scrambled siRNA transfection. (E and G) Extracellular flux analyzer-based Mito Stress test is performed in response to MDiVi-1 treatment (14μm) for 24 h in SKOV3 (n =4 ) and OAW42 (n =4 ) cells, respectively. (F and H) Calculated changes in basal respiration are represented as histogram. Data are expressed in ±SEM, and statistical significance was calculated using two-tailed Student’s t test (A) and one-way ANOVA (followed by Tukey post hoc analysis) (C-D, F, H) (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). NS stands for non-significant, also see Figures S6 and .

Article Snippet: siRNA for Ets1 , Santa Cruz , Sc-29309.

Techniques: Over Expression, Transfection, Two Tailed Test

Journal: iScience

Article Title: Ets1 facilitates EMT/invasion through Drp1-mediated mitochondrial fragmentation in ovarian cancer

doi: 10.1016/j.isci.2023.107537

Figure Lengend Snippet: Ets1-mediated metastasis/EMT is mitigated on Drp1 inhibition in ovarian cancer cells (A) Matrigel invasion assay is performed for Drp1 inhibition in SKOV3 cells (n =2 ). (B) Number of invading cells were quantitated and depicted as histogram. (C) Western blot analysis for Vimentin and N-Cadherin proteins on Drp1 inhibition is performed (n =4 ). α-tubulin serves as loading control. Densitometric quantitation is performed using ImageJ software and represented as histogram. (D) Immunocytochemistry-based microscopic analysis for E-Cadherin protein on MDiVi-1 (14μm) treatment is done in SKOV3 cells. (E) Fluorescence intensity quantitation is performed on LASX software and depicted as histogram. (F) Rhodamine-phalloidin-based microscopic imaging for F-Actin (RED) formation is performed on Drp1 silencing and inhibition (MDiVi-1 treatment) in SKOV3 cells. Yellow arrows indicate invading lamellipodia formation. Data are expressed in ±SEM, and statistical significance was calculated using one-way ANOVA (followed by Tukey post hoc analysis) (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). NS stands for non-significant. Scale bars 100 μM (A), 20 μM (D and F), also see Figure S8 .

Article Snippet: siRNA for Ets1 , Santa Cruz , Sc-29309.

Techniques: Inhibition, Invasion Assay, Western Blot, Control, Quantitation Assay, Software, Immunocytochemistry, Fluorescence, Imaging

Journal: iScience

Article Title: Ets1 facilitates EMT/invasion through Drp1-mediated mitochondrial fragmentation in ovarian cancer

doi: 10.1016/j.isci.2023.107537

Figure Lengend Snippet: MDiVi-1-mediated pharmacological inhibition of Drp1 mitigates EMT/invasion in in vivo syngeneic mice model (A) Representative images of tumors formed in C57BL/6 mice injected with ID8 cells and treated with PBS (Control) or MDiVi-1. (B) Relative tumor weight in control set and MDiVi-1 treated set. (C) Normalized tumor weight (mg) to total body weight (g) in control set and MDiVi-1-treated set. (D) Immunohistochemistry for PCNA and N-Cadherin expression in tumors on MDiVi-1 treatment with respect to control set. (E and F) Relative mean fluorescence intensity for PCNA and N-Cadherin proteins are depicted as scatterplots. (G) Immunohistochemistry for E-Cadherin and Vimentin expression in tumor sets. (H and I) Relative mean fluorescence intensity for E-Cadherin and Vimentin are depicted as scatterplots. (J) Schematic representation of Ets1-mediated Drp1 augmentation through direct binding at DNM1L promoter. Increased Drp1 undergoes activating phosphorylation at ser616 residue and enhanced mitochondrial recruitment to facilitate fission. Fragmented mitochondria due to pronounced fission lead to glycolytic bias over OXPHOS, resulting in EMT/invasion. MDiVi-1 administration blocks Drp1 activity ultimately to mitigate mitochondrial fission, glycolytic bias, and EMT/Invasion associated with Ets1. Data are expressed in ±SEM, and statistical significance was calculated using two-tailed Student’s t test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). NS stands for non-significant. Scale bars 50μM (C., F). Also see Figure S8 .

Article Snippet: siRNA for Ets1 , Santa Cruz , Sc-29309.

Techniques: Inhibition, In Vivo, Injection, Control, Immunohistochemistry, Expressing, Fluorescence, Binding Assay, Phospho-proteomics, Residue, Activity Assay, Two Tailed Test

Journal: iScience

Article Title: Ets1 facilitates EMT/invasion through Drp1-mediated mitochondrial fragmentation in ovarian cancer

doi: 10.1016/j.isci.2023.107537

Figure Lengend Snippet:

Article Snippet: siRNA for Ets1 , Santa Cruz , Sc-29309.

Techniques: Recombinant, Transfection, Western Blot, Membrane, cDNA Synthesis, Isolation, SYBR Green Assay, Chromatin Immunoprecipitation, Over Expression, Plasmid Preparation, Software

Journal: International Journal of Molecular Sciences

Article Title: Autoantibodies Targeting AT 1 - and ET A -Receptors Link Endothelial Proliferation and Coagulation via Ets-1 Transcription Factor

doi: 10.3390/ijms23010244

Figure Lengend Snippet: SRC-IgG activate Ets-1. Non-stimulated cells (Ctrl) were used as references when natural ligands were included, whereas Ctrl-IgG served as references when only IgG were used. ( a ) HMEC-1 were stimulated with AT-II, ET-1, Ctrl-IgG or SRC-IgG, with or without pre-incubation with MEK-1 inhibitor. ERK1/2 activation was measured as the pERK/α-Tubulin ratios. ( b ) Ets-1 transcriptional ( left ) and translational levels ( right ) were measured over time after stimulation with Ctrl- or SRC-IgG. ( c ) Specificity was asserted by pre-treatment with an AT 1 R or ET A R inhibitor (Valsartan or Sitaxentan, respectively), before stimulation with Ctrl- or SRC-IgG. ( d ) ( left and right ) HMEC-1 were incubated with Ctrl-IgG, natural ligands or SRC-IgG with or without pre-incubation with respective receptor blockers. ( a – d ) n = 4; representative blots are shown. * p < 0.05.

Article Snippet: Membranes were probed with phospho-ERK (Cell Signaling, Danvers, MA, USA), α-Tubulin (Sigma Aldrich, Saint Louis, MI, USA), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz, Dallas, TX, USA or Hytest Ltd., Turku, Finland), p38-Ets-1 (Invitrogen, Carlsbad, CA, USA), Ets-1 (Santa Cruz, Dallas, TX, USA), TF (American Diagnostica, Pfungstadt, Germany) antibodies. shRNA vectors provided by Santa Cruz were used to deliver scrambled or Ets-1 shRNA.

Techniques: Incubation, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Autoantibodies Targeting AT 1 - and ET A -Receptors Link Endothelial Proliferation and Coagulation via Ets-1 Transcription Factor

doi: 10.3390/ijms23010244

Figure Lengend Snippet: Endothelial cell proliferation elicited by SRC-IgG via ERK1/2–Ets-1 signaling. Non-stimulated cells (Ctrl) were used as reference when natural ligands were included, whereas Ctrl-IgG served as reference when only IgG were used. HMEC-1 were stimulated for 24 h with either natural ligands, Ctrl- or SRC-IgG, and specificity was assessed via two-hour pre-incubation with corresponding receptor inhibitors ( a ) ( left and right ) or cRaf1 inhibitor ( b ). ( c ) Abolition of Ets-1 translational regulation by shRNA following six-hour HMEC-1 stimulation. Ctrl shRNA corresponds to a mix of three control shRNA plasmids. Blots were over-exposed to better appreciate the decrease in the protein level. ( d ) Decrease in SRC-IgG induced endothelial cell proliferation by Ets-1 knockdown. ( a – c ) n = 4, ( d ) 7 ≤ n ≤ 11; representative blots are shown. * p < 0.05.

Article Snippet: Membranes were probed with phospho-ERK (Cell Signaling, Danvers, MA, USA), α-Tubulin (Sigma Aldrich, Saint Louis, MI, USA), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz, Dallas, TX, USA or Hytest Ltd., Turku, Finland), p38-Ets-1 (Invitrogen, Carlsbad, CA, USA), Ets-1 (Santa Cruz, Dallas, TX, USA), TF (American Diagnostica, Pfungstadt, Germany) antibodies. shRNA vectors provided by Santa Cruz were used to deliver scrambled or Ets-1 shRNA.

Techniques: Incubation, shRNA

Journal: International Journal of Molecular Sciences

Article Title: Autoantibodies Targeting AT 1 - and ET A -Receptors Link Endothelial Proliferation and Coagulation via Ets-1 Transcription Factor

doi: 10.3390/ijms23010244

Figure Lengend Snippet: Ets-1 binding to the TF promoter upon AT 1 R/ET A R stimulation by either respective natural peptide ligand or in response to SRC-IgG. ( a ) ( left and right ) Dual luciferase assay shows a TF promoter activity increase in response to either receptor-activating scenarios as compared with non-stimulated or Ctrl-IgG treated cells. ( b ) ( left and right ) Observed activation is abolished by specific AT 1 R or ET A R inhibitors. ( c ) EMSA performed with nucleus proteins of endothelial cells incubated with TF promoter DNA. Shift specificity was assessed using non-labeled DNA, the incubation with Ets-1-specific antibodies triggering a supershift. ( d ) ( left and right ) Chromatin immunoprecipitation (ChIP) performed using stimulated cells, the DNA of which was precipitated with an antibody directed against Ets-1. ( a ) left, ( b , d ) n = 4, ( a ) right, ( c ) n = 3; representative blots are shown. * p < 0.05.

Article Snippet: Membranes were probed with phospho-ERK (Cell Signaling, Danvers, MA, USA), α-Tubulin (Sigma Aldrich, Saint Louis, MI, USA), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz, Dallas, TX, USA or Hytest Ltd., Turku, Finland), p38-Ets-1 (Invitrogen, Carlsbad, CA, USA), Ets-1 (Santa Cruz, Dallas, TX, USA), TF (American Diagnostica, Pfungstadt, Germany) antibodies. shRNA vectors provided by Santa Cruz were used to deliver scrambled or Ets-1 shRNA.

Techniques: Binding Assay, Luciferase, Activity Assay, Activation Assay, Incubation, Labeling, Chromatin Immunoprecipitation

Journal: International Journal of Molecular Sciences

Article Title: Autoantibodies Targeting AT 1 - and ET A -Receptors Link Endothelial Proliferation and Coagulation via Ets-1 Transcription Factor

doi: 10.3390/ijms23010244

Figure Lengend Snippet: TF involvement in endothelial cell proliferation. Non-stimulated cells (Ctrl) were used as a reference when natural ligands were included, whereas Ctrl-IgG served as a reference when only IgG were used. ( a ) Transcriptional ( left ) and translational analysis ( right ) of TF after endothelial cell stimulation. ( b ) Specific inhibition of AT 1 R/ET A R abolishes TF activity increase. ( c ) Ets-1 knockdown abolishes TF protein synthesis. ( d ) BrdU incorporation shows that pre-incubation with a TF-blocking antibody annihilates endothelial cell proliferation elicited by SRC-IgG. ( a , c ) n = 4, ( d ), 4 ≤ n ≤ 7; representative blots are shown. * p < 0.05, ** p < 0.01.

Article Snippet: Membranes were probed with phospho-ERK (Cell Signaling, Danvers, MA, USA), α-Tubulin (Sigma Aldrich, Saint Louis, MI, USA), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz, Dallas, TX, USA or Hytest Ltd., Turku, Finland), p38-Ets-1 (Invitrogen, Carlsbad, CA, USA), Ets-1 (Santa Cruz, Dallas, TX, USA), TF (American Diagnostica, Pfungstadt, Germany) antibodies. shRNA vectors provided by Santa Cruz were used to deliver scrambled or Ets-1 shRNA.

Techniques: Cell Stimulation, Inhibition, Activity Assay, BrdU Incorporation Assay, Incubation, Blocking Assay

Journal: International Journal of Molecular Sciences

Article Title: Autoantibodies Targeting AT 1 - and ET A -Receptors Link Endothelial Proliferation and Coagulation via Ets-1 Transcription Factor

doi: 10.3390/ijms23010244

Figure Lengend Snippet: Proposed intracellular cascade following AT 1 R and ET A R activation by SRC-IgG. Binding of SRC-IgG to the receptors triggers the activation of cRaf1, MEK, ERK1/2 and, in turn, of Ets-1, through phosphorylation of its Thr38. Once activated, Ets-1 binds to the promoter of TF, triggering its expression (mRNA and protein). This intracellular pathway results in endothelial cell proliferation, inducing obliterative vasculopathy in SSc patients.

Article Snippet: Membranes were probed with phospho-ERK (Cell Signaling, Danvers, MA, USA), α-Tubulin (Sigma Aldrich, Saint Louis, MI, USA), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz, Dallas, TX, USA or Hytest Ltd., Turku, Finland), p38-Ets-1 (Invitrogen, Carlsbad, CA, USA), Ets-1 (Santa Cruz, Dallas, TX, USA), TF (American Diagnostica, Pfungstadt, Germany) antibodies. shRNA vectors provided by Santa Cruz were used to deliver scrambled or Ets-1 shRNA.

Techniques: Activation Assay, Binding Assay, Expressing