Journal: Oncogenesis

Article Title: Membrane-bound β-catenin degradation is enhanced by ETS2-mediated Siah1 induction in Helicobacter pylori -infected gastric cancer cells

doi: 10.1038/oncsis.2017.26

Figure Lengend Snippet: ETS2 binds to EBS in the 5′ UTR and induces siah1 transcription and protein expression in the H. pylori -infected GCCs. ( a ) Promoter and 5′ UTR analysis of human siah1 gene shows that an EBS located between +92 and +95 (represented by a box). We assume that the most upstream exon 1 of the Siah1 cDNA is at position +1 22 . ( b ) Western blot results showing the status of ETS2 binding with the siah1 5′ UTR ( n =3) in the presence or absence of H. pylori . ETS2 binds to the WT EBS only but not with the EBS-Mut oligo. Western blot of nuclear lysates shows the levels of ETS2 protein expression in the input lanes. HDAC1 is the loading control for nuclear lysates. ( c ) ChIP assay of ETS2 immunocomplex for siah1 EBS. IgG= immunoglobulin G; M= MW marker; NS= non-specific primer, S= specific primer. ( d ) Figure shows dual luciferase assay involving WT and ETS2-Mut siah1 5′ UTR-transfected and infected or uninfected MKN45 cells. Data are analyzed by two-way ANOVA with Tukey’s post hoc test ( n =3). Error Bars, s.e.m. *** P < 0.001, ** P < 0.01, * P < 0.05. ( e ) Bar graph of dual luciferase assay result showing transcriptional activation of WT siah1 5’ UTR with ectopic ETS2 expression and H. pylori infection. Data are analyzed by two-way ANOVA with Tukey’spost hoc test. Error bars, s.e.m. *** P <0.003; **** P< 0.0001. ( f ) Transient transfection of ETS2 siRNA followed by western blotting shows Siah1 suppression in the ETS2-suppressed MKN45 cells.

Article Snippet: ETS2 construct (#28128) was procured from Addgene, MA, USA.

Techniques: Expressing, Infection, Western Blot, Binding Assay, Control, Marker, Luciferase, Transfection, Activation Assay

Journal: Oncogenesis

Article Title: Membrane-bound β-catenin degradation is enhanced by ETS2-mediated Siah1 induction in Helicobacter pylori -infected gastric cancer cells

doi: 10.1038/oncsis.2017.26

Figure Lengend Snippet: Parallel ETS2 and Siah1 induction occurs in H. pylori- infected GCCs, as well as in human gastric adenocarcinoma biopsy samples. ( a ) A representative western blot ( n =3) showing optimal induction of ETS2 and Siah1 by 200 MOI of H. pylori at 3 h and 6 h p.i. ( b ) Cells infected for various time periods are analyzed for ETS2 and Siah1 protein expression ( n =3). ( c ) Western blot ( n =4 showing ETS2 and Siah1 proteins in uninfected and H. pylori -infected Kato III, MKN45 and AGS cells. ( d ) Western blot results ( n =3) depicting equal effectiveness of 8-1 and 26695 in inducing ETS2 and Siah1 proteins. Graphical representations of panels A–D are shown in .

Article Snippet: ETS2 construct (#28128) was procured from Addgene, MA, USA.

Techniques: Infection, Western Blot, Expressing

Journal: Oncogenesis

Article Title: Membrane-bound β-catenin degradation is enhanced by ETS2-mediated Siah1 induction in Helicobacter pylori -infected gastric cancer cells

doi: 10.1038/oncsis.2017.26

Figure Lengend Snippet: Induced ETS2 and Siah1 expression in Helicobacter -infected human and mouse gastric epithelia. ( a ) H&E staining of human non-cancer (a) and adenocarcinoma (e) biopsy samples ( n =10 for each group) and fluorescence microscopy of the staining for Siah1 (b and f), ETS2 (c and g) and DAPI (d and h). Original magnification × 100, inset × 400. Scales shown 50 μm. Inset scale 20 μm. ( b ) H&E staining of uninfected ( n =16) and infected ( n =16) antral gastric tissues from C57BL/6 mice (a and e, respectively) and their corresponding fluorescence microscopy images showing Siah1 (b and f), ETS2 (c and g) and DAPI (d and h) staining. Infected mice show inflammation (thin arrow), mucus gland metaplasia (open arrow) in the mucosa. ( c ) Data representing similar observations in another set of uninfected and infected mice gastric tissues. Original magnification × 100, inset × 400. Scales shown in b and c : 50 μm.

Article Snippet: ETS2 construct (#28128) was procured from Addgene, MA, USA.

Techniques: Expressing, Infection, Staining, Fluorescence, Microscopy

Journal: Oncogenesis

Article Title: Membrane-bound β-catenin degradation is enhanced by ETS2-mediated Siah1 induction in Helicobacter pylori -infected gastric cancer cells

doi: 10.1038/oncsis.2017.26

Figure Lengend Snippet: Siah1 promotes membrane-bound β-catenin degradation in H. pylori -infected GCCs. ( a ) Western blotting result ( n =3) of whole cell lysates prepared from Siah1 or empty vector (pcDNA3.1 + )-transfected and H. pylori -infected or uninfected MKN45 cells. Blots are incubated with Siah1 and β-catenin primary antibodies. α-tubulin is the loading control. ( b ) Decreased membrane-bound β-catenin expression is detected by western blotting ( n =3) in Siah1-overexpressed and H. pylori -infected cells. ( c ) Western blotting result ( n =3) of whole cell lysates prepared from siSiah1 or siControl duplex-transfected and H. pylori -infected or uninfected MKN45 cells. Blots are incubated with Siah1 and β-catenin primary antibodies. α-tubulin is the loading control. ( d ) Decreased membrane-bound β-catenin expression is seen in Siah1-supressed and H. pylori -infected cells. ( e ) Western blotting result ( n =3) of whole cell lysates prepared from siETS2 or siControl duplex-transfected and H. pylori -infected MKN45 cells. Blots are incubated with Siah1 and β-catenin primary antibodies. α-tubulin is the loading control. ( f ) Decreased membrane-bound β-catenin expression is detected in western blotting ( n =3) in ETS2-supressed and H. pylori -infected cells. ( g ) Siah1 is immunoprecipitated with anti-Siah1 antibody from the membrane fraction and immunoblotted to detect β-catenin and Siah1 interaction. Non-specific band=immunoglobulin heavy chain. Graphical representations are shown in .

Article Snippet: ETS2 construct (#28128) was procured from Addgene, MA, USA.

Techniques: Membrane, Infection, Western Blot, Plasmid Preparation, Transfection, Incubation, Control, Expressing, Immunoprecipitation

Journal: Oncogenesis

Article Title: Membrane-bound β-catenin degradation is enhanced by ETS2-mediated Siah1 induction in Helicobacter pylori -infected gastric cancer cells

doi: 10.1038/oncsis.2017.26

Figure Lengend Snippet: Siah1 increases the rate of cell migration in H. pylori -infected GCCs. ( a ) Graphical representations ( n =3) of wound healing assay showing enhanced migration potential of Siah1-overexpressed and H. pylori -infected cells. 24 h post transfection, wound is marked and the scratched area is monitored from 6 h-24 h. Data have been analyzed by 2-way ANOVA with Tukey’s post hoc test. Error bars, s.e.m. **** P <0.0001. ( b ) Cell migration assay performed in transwell chambers with AGS cells show decreased migration of infected cells expressing siSiah1 as compared to the siControl group. Arrowheads indicate migrated cells; scales shown: 50 μm. Bar graphs denote the average number of migrated cells (mean±s.e.m., n =3). Data are analyzed by 2-way ANOVA with Tukey’s post hoc test. Error bars, s.e.m. *** P <0.003; **** P <0.0001. Protein level of siSiah1 cells are shown in the accompanying western blot images. ( c ) Matrigel invasion assay with Siah1-suppressed AGS cells showing reduced invasiveness in H. pylori -infected GCCs. Bar graphs denote the average number of cells invaded through the Transwell matrigel ( n =3). Arrowheads indicate invaded cells. Data are analyzed by two-way ANOVA with Tukey’s post hoc test. Error bars, s.e.m. ** P <0.01; *** P <0.003; **** P <0.0001. Scales shown: 50 μm. ( d ) Soft agar colony formation assay is performed on MKN45 cells. Siah1 stably-transfected cells show a substantial increase in colony forming ability post H. pylori infection as compared to cells expressing the empty vector. Siah1 level in Siah1-stable cells are shown in the accompanying western blot result. ( e ) Soft agar assay performed with uninfected or infected MKN45 cells that stably-express either ETS2 or empty vector show a substantial increase in colony forming ability of ETS2-expressing cells. Protein level of ETS2 stable cells are shown in the accompanying western blot images. Scales shown in b and c : 100 μm.

Article Snippet: ETS2 construct (#28128) was procured from Addgene, MA, USA.

Techniques: Migration, Infection, Wound Healing Assay, Transfection, Cell Migration Assay, Expressing, Western Blot, Invasion Assay, Soft Agar Assay, Stable Transfection, Plasmid Preparation

Journal: Circulation Research

Article Title: Ets2 Determines the Inflammatory State of Endothelial Cells in Advanced Atherosclerotic Lesions

doi: 10.1161/circresaha.111.243444

Figure Lengend Snippet: Figure 1A), but mean Ets2 levels do not differ between stable fibrous or vulnerable atheromatous plaque phenotype (P0.18; Figure 1B). Ets2 protein levels specifically corre- lated with increased plaque thrombogenicity and erythrocyte extravasation in atheromatous plaques (P0,05 versus Q2, Figure 1C), the phenotypic characteristics of vulnerable plaque. In contrast, these correlations were absent in fibrous atherosclerotic lesions (Figure 1D). To further investigate the relation between Ets2 levels and inflammation in human atherosclerotic lesions, tumor necro- sis factor- (TNF), IL6, and monocyte chemoattractant protein 1 (MCP1) levels were assessed using commercial ELISAs. Ets2 protein levels showed correlations with TNF, IL6, and MCP1 levels (TNF: R0.31, P0.03; IL6: R0.31, P0.04; and MCP1: R0.40; P0.006; Figure 2A through 2C). In contrast, Ets2 showed an inverse correlation with VEGF-A protein levels (R0.22; P0.05, Figure 2D). Immunohistological analysis of serial sections shows specific expression of Ets2 in the neocapillaries of human advanced atherosclerotic plaques and in the endothelium covering the lesion (Figure 2E). Taken together, these find- ings point toward a possible regulatory role for Ets2 in angiogenesis, vascular integrity, and inflammatory state in vulnerable plaque.

Article Snippet: For western blot analysis, cell culture and tissue samples were lysed in NP40 buffer, and analyzed using SDS-PAGE western blots and 1:1000 rabbit polyclonal antibody against Ets2 (Aviva Systems, the Supplement Material 7 Netherlands).

Techniques: Expressing

Journal: Circulation Research

Article Title: Ets2 Determines the Inflammatory State of Endothelial Cells in Advanced Atherosclerotic Lesions

doi: 10.1161/circresaha.111.243444

Figure Lengend Snippet: Figure 2. Rising Ets2 levels in human atherosclerotic lesions correlate with MCP1, TNF, and IL6 and are inversely correlated with VEGFA. Posi- tive correlations were observed between Ets2 and A, TNF (R0.31; P0.03); B, IL6 (R0.31; P0.04); and C, MCP1 (R0.40; P0.006) in human carotid endarterectomy samples. D, VEGFA lev- els showed a negatively correlation with Ets2 (Pearson correlation, R0.22; P0.05). E, Immunohistological staining of serial sections shows predominant expression of Ets2 in CD31 neocapil- laries in advanced human atherosclerotic lesion. First 2 panels on the left show the Ets2 and CD31 signal in the intimal neovasculature, whereas the last panel shows Ets2 and CD31 expression in the endothelium covering the atherosclerotic lesion. Primary antibodies were detected using a horseradish peroxidase–labeled secondary antibody, followed by nickel- DAP detection (brown) and eosin coun- terstaining. Immunostaining using matched IgG control on the same mate- rial shows limited background staining for the secondary antibody.

Article Snippet: For western blot analysis, cell culture and tissue samples were lysed in NP40 buffer, and analyzed using SDS-PAGE western blots and 1:1000 rabbit polyclonal antibody against Ets2 (Aviva Systems, the Supplement Material 7 Netherlands).

Techniques: Staining, Expressing, Labeling, Immunostaining, Control

Journal: Circulation Research

Article Title: Ets2 Determines the Inflammatory State of Endothelial Cells in Advanced Atherosclerotic Lesions

doi: 10.1161/circresaha.111.243444

Figure Lengend Snippet: Figure 3. Endogenous expression of Ets2 in ApoE/ mice. A, Endothelial Ets2 expression in aortic segments of C57bl/6 ApoE/ mice that received normal chow (ApoE/ chow) versus cholesterol-rich, high-fat diet for 3 weeks (ApoE/ Western Diet [WD]), as assessed by whole-mount en face immunostaining and subsequent visualization of the samples using fluores- cent confocal microscopy. Red fluores- cence indicates the Ets2 immunofluores- cent signal, and green fluorescence is due to autofluorescence of the basal elastic lamina. Representative samples of one experiment with 5 animals per group are shown. B, qPCR analysis showed en- dogenous Ets2 mRNA level in lesions with the vulnerable phenotype, compared with the contralateral naive carotid arteries and stable plaques in a mouse ApoE/-based model of VP at week 9 (n6 per group; *P0.05 versus control. †P0.05 versus stable). C, Cross-sectional immunohisto- logical analysis in carotid vessels derived from ApoE/ animals on normal chow diet showed very weak Ets2 staining. High magnification of the endothelium demon- strated that intracellular Ets2 was mainly located outside the cell nuclei. Areas of nuclei are marked with an open circle. D, Ets2 expression was clearly detected in CD31 ECs in murine VP lesions and colocalized with nuclear DAPI staining (indicated by white arrows). E, In contrast colocalization of Ets2 (red fluorescent sig- nal) with CD68 (green fluorescent signal) macrophages in the lesion was limited. Dotted white lines mark the boundary between intima and media. indicates lumen area.

Article Snippet: For western blot analysis, cell culture and tissue samples were lysed in NP40 buffer, and analyzed using SDS-PAGE western blots and 1:1000 rabbit polyclonal antibody against Ets2 (Aviva Systems, the Supplement Material 7 Netherlands).

Techniques: Expressing, Western Blot, Immunostaining, Confocal Microscopy, Control, Derivative Assay, Staining

Journal: Circulation Research

Article Title: Ets2 Determines the Inflammatory State of Endothelial Cells in Advanced Atherosclerotic Lesions

doi: 10.1161/circresaha.111.243444

Figure Lengend Snippet: Figure 4. Overexpression of Ets2 destabilizes VP. Morphometric and immunohistological analysis of VPs in ApoE/ mice that were transfected with E1-Ad or Ets2-Ad, 9 weeks after VP induction. A through C, In the right column, representative cross- sections are shown. In the left column, bar graphs show the effects of Ets2 transduction on A, intima/ media (I/M) ratio; B, % necrotic core (necrotic core area/intima area*100%); and C, relative cap thick- ness (ie, mean cap thickness at plaque shoulders and midregion/ maximal intimal thickness) in vulnera- ble lesions of E1-Ad, as compared with Ets2-Ad– transfected ApoE/ mice. D through G, Morpho- metric and immunohistological analysis of VPs in E1-Ad–transfected or Ets2-Ad–transfected ApoE/

Article Snippet: For western blot analysis, cell culture and tissue samples were lysed in NP40 buffer, and analyzed using SDS-PAGE western blots and 1:1000 rabbit polyclonal antibody against Ets2 (Aviva Systems, the Supplement Material 7 Netherlands).

Techniques: Over Expression, Transfection, Transduction

Journal: Circulation Research

Article Title: Ets2 Determines the Inflammatory State of Endothelial Cells in Advanced Atherosclerotic Lesions

doi: 10.1161/circresaha.111.243444

Figure Lengend Snippet: Figure 5. Ets2 expression promotes new vessel formation and intraplaque hemorrhaging in vulnerable plaque. Immuno- histological analysis of VPs in ApoE/ mice that were trans- fected with E1-Ad or Ets2-Ad, 9 weeks after VP induction. A, Representative cross sections and bar graphs show the effects of Ets2 transduction on the % of adventitial CD31 ECs. In the photograph panel, the green fluorescent signal represents auto- fluorescence from the elastic lamina, and the white triangle symbol indicates the adventitial side (n10 for each group; *P0,05 versus E1-Ad). B, Micrographs show representative immunohistological staining of Ter119 (red) to identify IPH in the VP area. Nuclei are identified by DAPI (blue fluorescent signal). The green fluorescent signal represents the autofluorescence of the elastic laminas. Lumen boundaries are marked by a white dotted line. Bar graph shows the effect of adventitial Ets2 trans- duction on vascular integrity by scoring IPH defined as the % of Ter119 intimal areas (n10 for each group; *P0.05 versus E1-Ad), C, High-magnification micrographs of these murine lesions clearly identify Ter119 (red fluorescent signal) erythro- cytes located outside CD31 vascular structures (green fluores- cent signal) as a result of erythrocyte extravasation during intra- plaque hemorrhaging. The white open arrow points out an intimal microvessel with open lumen.

Article Snippet: For western blot analysis, cell culture and tissue samples were lysed in NP40 buffer, and analyzed using SDS-PAGE western blots and 1:1000 rabbit polyclonal antibody against Ets2 (Aviva Systems, the Supplement Material 7 Netherlands).

Techniques: Expressing, Transduction, Staining

Journal: Circulation Research

Article Title: Ets2 Determines the Inflammatory State of Endothelial Cells in Advanced Atherosclerotic Lesions

doi: 10.1161/circresaha.111.243444

Figure Lengend Snippet: Figure 6. Ets2 responds to TNF stimulation and promotes gene expression of angiogenic regulators in ECs. A, Endogenous Ets2 mRNA levels in HUVECs were not responsive to different doses of LDL (range, 0 to 50 g/mL), or B, oxLDL (range, 0 to 50 g/mL) as assessed by qPCR at different time points. C, Similarly hypoxia failed to induce Ets2 expression (normal oxygen, 21% O2 versus low oxygen 3% O2). D, TNF stimulation (range, 0 to 50 ng/mL) induced a dose-response in Ets2 mRNA expression (n4; *P0,05 versus 0 ng/mL TNF); E, total Ets2 protein levels (n3; *P0,05 versus 0 ng/mL TNF); and F, Ets2 phosphorylation at the Thr72 activation site (n3; *P0,05 versus 0 ng/mL TNF) after 24-hour stimulation. G, Stimulation of HUVECs with 25 ng/mL TNF for 24 hours triggered translocation of Ets2 from the perinuclear compartment to the nucleus, as demonstrated by immunofluorescent staining and subsequent analysis by confocal microscopy. Shown in the figure are representative samples of 3 different experiments. Red indicates the Ets2 immunofluorescent signal. H, qPCR analysis of HUVECs transfected with Ets2 targeting siRNA showed reduc- tion in expression levels of proangiogenic genes, including HO-1, EphB4, EphrinB2, DLL4, and Jagged1, whereas expression of VEGFA and VEGFR2 were not affected. In addition, angiopoietin (Ang) 2 was downregulated by Ets2 silencing, whereas Ang 1was not affected (n5, *0.05 versus sisham).

Article Snippet: For western blot analysis, cell culture and tissue samples were lysed in NP40 buffer, and analyzed using SDS-PAGE western blots and 1:1000 rabbit polyclonal antibody against Ets2 (Aviva Systems, the Supplement Material 7 Netherlands).

Techniques: Gene Expression, Expressing, Phospho-proteomics, Activation Assay, Translocation Assay, Staining, Confocal Microscopy, Transfection

Journal: Circulation Research

Article Title: Ets2 Determines the Inflammatory State of Endothelial Cells in Advanced Atherosclerotic Lesions

doi: 10.1161/circresaha.111.243444

Figure Lengend Snippet: Figure 7. Ets2 regulates expression of proinflammatory cytokines and adhesion molecules and compromises endothelial integ- rity. qPCR analysis of MCP1, IL6, VCAM1, and ICAM expression in response to Ets2 silencing at basal levels and in response to TNF stimulation after 24 hours. SiRNA-mediated silencing of Ets2 inhibited expression of A, MCP1; B, IL6; and C, VCAM1, whereas D, ICAM1 expression, was not affected, as compared with nontargeting scrambled siRNA transfected controls (sisham). A through D, Stimulation with 25 ng/mL TNF increased the expression of MCP1, IL6, VCAM1, and ICAM1. Ets2 silencing similarly inhibited the response of MCP1, IL6, and VCAM1 to TNF stimulation, whereas ICAM1 remained unaffected. E, Basal IL6, and F, MCP1 protein lev- els in the supernatant were decreased by Ets2 silencing, as demonstrated in dedicated ELISA assays. Stimulation with 25 ng/mL TNF increased IL6 and MCP1 protein levels, and Ets2 silencing inhibited the response of IL6 and MCP1 to TNF (qPCR data, n4, ELISA data, n6; *P0.05 versus sisham or sishamTNF). Ets2 overexpression resulted in loss of endothelial integrity. G, Measurement of Dextran-FITC leakage through a monolayer of E1-Ad or Ets2-Ad transfected HUVECs with or without TNF stimulation. Graphs show mean intensity of fluorescence (Y-axes) of dextran-FITC measured in the lower compartment in response to 25 ng/mL TNF in minutes (X-axes). Black bars indicate the response of E1-Ad and white bars indicate Ets2-Ad transfected HUVEC monolayers (n3, #P0.1 versus E1-Ad, *P0.05 versus E1-Ad). H, qPCR analysis of ADAM10 expression in response to Ets2 overexpression in HUVECs at basal levels and after 24-hour stimulation of 25 ng/mL TNF (n4 experiments, *P0.05 versus E1-Ad and E1-AdTNF, ‡P0,05 versus Ets2-Ad). Ets2 overexpression in the retina of ApoE/ mice promoted vascular leakage. I, Three days after intravitreal injection

Article Snippet: For western blot analysis, cell culture and tissue samples were lysed in NP40 buffer, and analyzed using SDS-PAGE western blots and 1:1000 rabbit polyclonal antibody against Ets2 (Aviva Systems, the Supplement Material 7 Netherlands).

Techniques: Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Over Expression, Injection

Journal: Circulation Research

Article Title: Ets2 Determines the Inflammatory State of Endothelial Cells in Advanced Atherosclerotic Lesions

doi: 10.1161/circresaha.111.243444

Figure Lengend Snippet: Figure 8. Ets2 silencing by shRNA promotes vulnerable plaque stabilization. Morphometric and immunohistological analysis of VPs in ApoE/ mice that were transfected with a lentiviral construct that expresses Ets2 targeting shRNA (LV-siEts2) or a control lentiviral construct that expresses nontargeting shRNA (LV-sisham), 9 weeks after VP induction. A, qPCR assessment of endogenous Ets2 mRNA expression in LV-siEts2–transduced carotid vessel segments of ApoE/ mice as compared with LV-sisham–treated animals and untreated contralateral carotid arteries after 4 days of transfection (*P0.05 versus control and LV-sisham; n4 for qPCR assess- ment; *P0.05 versus control). B through J, Representative cross sections and bar graphs show the effects of shRNA-mediated silenc- ing of Ets2 on B, intima/media (I/M) ratio; C, % necrotic core (necrotic core area/intima area*100%); and D, relative cap thickness (ie, mean cap thickness at plaque shoulders and midregion/ maximal intimal thickness) in vulnerable lesions of LV-sisham, as compared with LV-siEts2 transfected ApoE/ mice. E through G, Analysis of plaque phenotype show the effects of Ets2 silencing on E, % intimal lipids; F, % intimal CD68 macrophages; G, % intimal VSMCs; and H, and % intimal collagen, in vulnerable lesions of LV-sisham as compared with LV-siEts2 transfected ApoE/ mice (n8 for each group; *P0.05 versus LV-sisham). In the photographs, dotted lines indicate the luminal area. In the micrographs that show VSMC-actin or CD68 staining, the green fluorescent signal represents the auto- fluorescence of the elastic lamina. Representative cross sections and bar graphs show the effects of Ets2 silencing using a lentiviral siRNA construct on vascularization and intraplaque hemorrhaging: I, The % of intimal CD31 ECs, and J, % of IPH in ApoE/ mice transfected with LV-sisham or LV-siEts2. Representative micrographs of CD31 and Ter119 staining are shown. For Ter119, nuclei are identified by DAPI (blue fluorescent signal), and lumen boundaries are marked by a white dotted line (n8 for each group; *P0.05 ver- sus LV-sisham).

Article Snippet: For western blot analysis, cell culture and tissue samples were lysed in NP40 buffer, and analyzed using SDS-PAGE western blots and 1:1000 rabbit polyclonal antibody against Ets2 (Aviva Systems, the Supplement Material 7 Netherlands).

Techniques: shRNA, Transfection, Construct, Control, Expressing, Staining

Journal: Cell Death & Disease

Article Title: Impact of N-acetyltransferase 10 on macrophage activation and inflammation-induced cardiac dysfunction

doi: 10.1038/s41419-025-07796-6

Figure Lengend Snippet: A Venn diagram showing the downstream target genes regulated by NAT10 via ac4C modification. Left: Hypoacetylated genes after NAT10 knockout (acRIP-seq); Right: Downregulated genes after NAT10 knockout (RNA-seq, P < 0.05, log2FoldChange < -1). B Fold changes in transcript levels (RNA-seq) and ac4C modification levels of 44 downstream targets. C Visualization of ac4C peaks in Ets2 across different groups using IGV software. D RT-qPCR detection of the relative enrichment of Ets2 mRNA in acRIP products from BMDMs. E RT-qPCR detection of the relative enrichment of Ets2 mRNA in NAT10 RIP products from BMDMs. F Decay curves for Ets2 mRNA in Flox and Nat10-/- BMDMs. G RT-qPCR detection of Ets2 mRNA expression in BMDMs after Nat10 knockout. H Volcano plot of differentially expressed genes ( | log2FC | > 1 and FDR < 0.05) at the translation level in LPS-treated BMDMs after Nat10 knockout. I Venn diagram showing the overlap of target genes with changes in ac4C acetylation, transcription, and translation levels after NAT10 knockout. J Heatmap of translation efficiency (Ribo-seq) for the overlapping 27 target genes., expressed as Z-score in relation to median. K KEGG analysis of differentially expressed genes (DEGs) in Ribo-seq. L Nat10-depleted or control cells were transfected with lentivirus containing pmirGLO-Ets2 reporter for 24 h, and Ets2 translation efficiency was defined as reporter protein production (F-luc/R-luc) divided by mRNA abundance. M Relative mRNA distribution of Ets2 in ribosome fractions analyzed by qRT-PCR in BMDMs. N Western blot analysis and densitometric quantification of ETS2 protein in Flox and Nat10-/- BMDMs. O Immunofluorescence analysis showing changes in ETS2 localization and expression. (scale bar = 10 um). Data are shown as mean ± SD. Statistical analyses were performed using the student t -test ( E , L ) and two-way two-sided ANOVA ( D , G , N ).

Article Snippet: The Ets2 or shRNA plasmid-containing lentivirus (Santa Cruz Biotechnology, sc-37856-V and sc-76850-V) was purchased and used as reported previously for lentiviral transduction of bone marrow-derived macrophages (BMDMs) seeded in six-well plates at a density of 1 × 106 cells per well following a five-day bone marrow cell culture.

Techniques: Modification, Knock-Out, RNA Sequencing, Software, Quantitative RT-PCR, Expressing, Control, Transfection, Western Blot, Immunofluorescence

Journal: Cell Death & Disease

Article Title: Impact of N-acetyltransferase 10 on macrophage activation and inflammation-induced cardiac dysfunction

doi: 10.1038/s41419-025-07796-6

Figure Lengend Snippet: A Western blot detection of iNOS, NAT10, and ETS2 protein levels in BMDMs. Flox and Nat10-/- BMDMs were transfected with or without Ets2 lentivirus. B– D Flow cytometry analysis of macrophage surface markers CD80 and CD86 expression in BMDMs of each group( n = 6 per group). E Real-time PCR analysis of mRNA expression of cytokines in BMDMs of each group( n = 6 per group). F ELISA analysis of cytokine secretion in the culture medium of BMDMs of each group( n = 6 per group). G Western blot detection of iNOS, NAT10, and ETS2 protein levels in RAW264.7 cells. WT and Nat10 stable overexpressing RAW264.7 cells were transfected with or without Ets2 lentivirus. H– J Flow cytometry analysis of macrophage surface markers CD80 and CD86 expression in RAW264.7 cells of each group( n = 6 per group). K Real-time PCR analysis of mRNA expression of cytokines in RAW264.7 cells of each group( n = 6 per group). L ELISA analysis of cytokine secretion in the culture medium of RAW264.7 cells of each group ( n = 6 per group). Data are shown as mean ± SD. Statistical analyses were performed using two-way two-sided ANOVA.

Article Snippet: The Ets2 or shRNA plasmid-containing lentivirus (Santa Cruz Biotechnology, sc-37856-V and sc-76850-V) was purchased and used as reported previously for lentiviral transduction of bone marrow-derived macrophages (BMDMs) seeded in six-well plates at a density of 1 × 106 cells per well following a five-day bone marrow cell culture.

Techniques: Western Blot, Transfection, Flow Cytometry, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay