Journal: International Journal of Immunopathology and Pharmacology

Article Title: ETS variant transcription factor 6 enhances oxidized low-density lipoprotein-induced inflammatory response in atherosclerotic macrophages via activating NF-κB signaling

doi: 10.1177/20587384221076472

Figure Lengend Snippet: Primer sequences.

Article Snippet: After incubation with 5% normal goat serum for 1 h at room temperature, the sections were incubated with rabbit anti-ETV6 antibody (1:200, PA5-109,697, Thermo Fisher Scientific) and rat anti-F4/80 (10 μg/ml, ab6640, Abcam) overnight at 4 o C. After three washes with PBS, the samples were incubated with 5 μg/ml Alexa Fluor® 488-conjugated goat anti-rat IgG H&L and Alexa Fluor® 594-conjugated goat anti-rabbit IgG H&L (1:1000, ab150157, ab150080, Abcam) for an hour at room temperature.

Techniques:

Journal: International Journal of Immunopathology and Pharmacology

Article Title: ETS variant transcription factor 6 enhances oxidized low-density lipoprotein-induced inflammatory response in atherosclerotic macrophages via activating NF-κB signaling

doi: 10.1177/20587384221076472

Figure Lengend Snippet: ETV6 expression in aorta macrophages . (a) FACS dot plots showing aorta macrophages and monocytes. Intact cells were gated within singlets, followed by gating CD3/CD19/Gr-1/CD11c/NK1.1 − CD11b + cells. Macrophages and monocytes were then recognized based on the expression of F4/80 and Ly-6C. WT: wild-type mice. ApoE: atherosclerotic ApoE −/- mice. Ma: macrophages. Mo: monocytes. (b) F4/80 + macrophages in mouse spleens. (c) ETV6 mRNA in aorta macrophages and monocytes. WT-S: wild-type mouse spleen. AP-S: ApoE −/- mouse spleen. WT-A: wild-type mouse aorta. AP-A: ApoE −/- mouse aorta. N = 6 mice. *, p <0.05; **, p <0.01. (d) ETV6 protein in each cell population. The data represent two independent immunoblotting tests.

Article Snippet: After incubation with 5% normal goat serum for 1 h at room temperature, the sections were incubated with rabbit anti-ETV6 antibody (1:200, PA5-109,697, Thermo Fisher Scientific) and rat anti-F4/80 (10 μg/ml, ab6640, Abcam) overnight at 4 o C. After three washes with PBS, the samples were incubated with 5 μg/ml Alexa Fluor® 488-conjugated goat anti-rat IgG H&L and Alexa Fluor® 594-conjugated goat anti-rabbit IgG H&L (1:1000, ab150157, ab150080, Abcam) for an hour at room temperature.

Techniques: Expressing, Western Blot

Journal: International Journal of Immunopathology and Pharmacology

Article Title: ETS variant transcription factor 6 enhances oxidized low-density lipoprotein-induced inflammatory response in atherosclerotic macrophages via activating NF-κB signaling

doi: 10.1177/20587384221076472

Figure Lengend Snippet: ETV6 expression in aorta macrophages after oxLDL and/or LPS treatment. (a) ETV6 mRNA in wild-type aorta macrophages after incubation with oxLDL and/or LPS. N=8 mice. (b) ETV6 protein in aorta macrophages. (c) ETV6 mRNA in aorta macrophages after treatment in the presence or absence of QNZ. (d) ETV6 protein in aorta macrophages after treatment in the presence or absence of QNZ. N = 6 samples. *, p <0.05; **, p <0.01; ***, p <0.001.

Article Snippet: After incubation with 5% normal goat serum for 1 h at room temperature, the sections were incubated with rabbit anti-ETV6 antibody (1:200, PA5-109,697, Thermo Fisher Scientific) and rat anti-F4/80 (10 μg/ml, ab6640, Abcam) overnight at 4 o C. After three washes with PBS, the samples were incubated with 5 μg/ml Alexa Fluor® 488-conjugated goat anti-rat IgG H&L and Alexa Fluor® 594-conjugated goat anti-rabbit IgG H&L (1:1000, ab150157, ab150080, Abcam) for an hour at room temperature.

Techniques: Expressing, Incubation

Journal: International Journal of Immunopathology and Pharmacology

Article Title: ETS variant transcription factor 6 enhances oxidized low-density lipoprotein-induced inflammatory response in atherosclerotic macrophages via activating NF-κB signaling

doi: 10.1177/20587384221076472

Figure Lengend Snippet: Lentivirus-mediated ETV6 knockdown. (a) FACS dot plots indicating GFP expression in non-transduced peritoneal macrophages and LSh-E-transduced peritoneal macrophages 2 days after transduction (before puromycin selection) and 4 days after transduction (2 days after puromycin selection). (b) Fluorescent microscopy showing GFP expression in macrophages. Original magnification = 100×. (c to g) The expression of indicated cytokines in macrophages 6 days after transduction. N = 6. (h) Cell death in macrophages. The data represent two independent FACS experiments.

Article Snippet: After incubation with 5% normal goat serum for 1 h at room temperature, the sections were incubated with rabbit anti-ETV6 antibody (1:200, PA5-109,697, Thermo Fisher Scientific) and rat anti-F4/80 (10 μg/ml, ab6640, Abcam) overnight at 4 o C. After three washes with PBS, the samples were incubated with 5 μg/ml Alexa Fluor® 488-conjugated goat anti-rat IgG H&L and Alexa Fluor® 594-conjugated goat anti-rabbit IgG H&L (1:1000, ab150157, ab150080, Abcam) for an hour at room temperature.

Techniques: Knockdown, Expressing, Transduction, Selection, Microscopy

Journal: International Journal of Immunopathology and Pharmacology

Article Title: ETS variant transcription factor 6 enhances oxidized low-density lipoprotein-induced inflammatory response in atherosclerotic macrophages via activating NF-κB signaling

doi: 10.1177/20587384221076472

Figure Lengend Snippet: Effect of ETV6 knockdown on oxLDL-induced inflammatory reaction in macrophages in vitro . (A and B) ETV6 protein in macrophages after vehicle or oxLDL treatment. Representative Immunoblotting images are shown in (a). Statistics are shown in (b). LSh-C: LSh-C-transduced macrophages. LSh-E: LSh-E-transduced macrophages. (c to e) The expression of indicated cytokines in macrophages. N = 6. *, p <0.05; **, p <0.01; ***, p <0.001. (F and G) FACS histograms indicating the uptake of oxLDL (f) and cholesterol (g) by macrophages. V: vehicle. D: DyLight 550 oxLDL. N: NBD cholesterol. The data represent two independent FACS experiments.

Article Snippet: After incubation with 5% normal goat serum for 1 h at room temperature, the sections were incubated with rabbit anti-ETV6 antibody (1:200, PA5-109,697, Thermo Fisher Scientific) and rat anti-F4/80 (10 μg/ml, ab6640, Abcam) overnight at 4 o C. After three washes with PBS, the samples were incubated with 5 μg/ml Alexa Fluor® 488-conjugated goat anti-rat IgG H&L and Alexa Fluor® 594-conjugated goat anti-rabbit IgG H&L (1:1000, ab150157, ab150080, Abcam) for an hour at room temperature.

Techniques: Knockdown, In Vitro, Western Blot, Expressing

Journal: International Journal of Immunopathology and Pharmacology

Article Title: ETS variant transcription factor 6 enhances oxidized low-density lipoprotein-induced inflammatory response in atherosclerotic macrophages via activating NF-κB signaling

doi: 10.1177/20587384221076472

Figure Lengend Snippet: Impact of ETV6 knockdown on oxLDL-induced NF-κB pathway. (A to C) Phosphorylation of IKKβ (Ser180) and NF-κB p65 (Ser536) in macrophages after oxLDL treatment. Immunoblotting images are shown in (a), and statistics are shown in (b) and (c), respectively. LSh-C: LSh-C-transduced macrophages. LSh-E: LSh-E-transduced macrophages. (D to F) Expression of IκBα and nuclear NF-κB p65 in macrophages. Immunoblotting images are shown in (d), and statistics of IκBα and nuclear NF-κB p65 levels are shown in (e and f), respectively. N = 5. (g) FACS histograms indicating the expression of active Caspase-1 in macrophages after oxLDL treatment. N = 6. *, p <0.05; ***, p <0.001.

Article Snippet: After incubation with 5% normal goat serum for 1 h at room temperature, the sections were incubated with rabbit anti-ETV6 antibody (1:200, PA5-109,697, Thermo Fisher Scientific) and rat anti-F4/80 (10 μg/ml, ab6640, Abcam) overnight at 4 o C. After three washes with PBS, the samples were incubated with 5 μg/ml Alexa Fluor® 488-conjugated goat anti-rat IgG H&L and Alexa Fluor® 594-conjugated goat anti-rabbit IgG H&L (1:1000, ab150157, ab150080, Abcam) for an hour at room temperature.

Techniques: Knockdown, Phospho-proteomics, Western Blot, Expressing

Journal: International Journal of Immunopathology and Pharmacology

Article Title: ETS variant transcription factor 6 enhances oxidized low-density lipoprotein-induced inflammatory response in atherosclerotic macrophages via activating NF-κB signaling

doi: 10.1177/20587384221076472

Figure Lengend Snippet: Effect of ETV6 knockdown in vivo . (A and B) FACS dot plots indicating recognizing and sorting exogenous GFP + macrophages from aorta mononuclear cells (a) and splenocytes (b) of recipient mice. LSh-C: mice receiving LSh-C-transduced macrophages. LSh-E: mice receiving LSh-E-transduced macrophages. (c) ETV6 mRNA in sorted exogenous GFP + macrophages. (d to h) The expression of indicated cytokines in sorted exogenous GFP + macrophages. N = 8 mice. *, p <0.05; ***, p <0.001.

Article Snippet: After incubation with 5% normal goat serum for 1 h at room temperature, the sections were incubated with rabbit anti-ETV6 antibody (1:200, PA5-109,697, Thermo Fisher Scientific) and rat anti-F4/80 (10 μg/ml, ab6640, Abcam) overnight at 4 o C. After three washes with PBS, the samples were incubated with 5 μg/ml Alexa Fluor® 488-conjugated goat anti-rat IgG H&L and Alexa Fluor® 594-conjugated goat anti-rabbit IgG H&L (1:1000, ab150157, ab150080, Abcam) for an hour at room temperature.

Techniques: Knockdown, In Vivo, Expressing