Journal: Cell & Bioscience

Article Title: Deficiency in steroid receptor coactivator 3 enhances cytokine production in IgE-stimulated mast cells and passive systemic anaphylaxis in mice

doi: 10.1186/2045-3701-4-21

Figure Lengend Snippet: Passive systemic anaphylaxis in wild-type and SRC-3 -/- mice. SRC-3 +/+ (n = 5) and SRC-3 -/- mice (n = 5) were sensitized with anti-DNP IgE and DNP-HSA to induced systemic anaphylaxis as described in methods. Passive systemic anaphylaxis was monitored by measuring rectal temperatures after DNP-HSA challenge. Data represent the mean rectal temperature ± SD. * p <0.05 versus SRC-3 +/+ mice by t-test.

Article Snippet: RPMI 1640, DMSO, sodium pyruvate, antibody against β-actin and flag, anti-DNP IgE (clone SPE-7) were obtained from Sigma Aldrich (Sigma, St Louis, MO, USA); DNP-human serum albumin (DNP-HSA) was purchased from Biosearch Technologies (Biosearch Technologies, Novato CA, USA); recombinant murine SCF and IL-3 were purchased from peprotech (Peprotech, Rocky Hill, NJ, USA); anti-mouse CD117 (c-Kit)-PE, anti-mouse FcϵRI-FITC, IL-6 ELISA kit, and TNF-α ELISA kit were obtained from eBioscence (eBioscence, San Diego, CA, USA); nonessential amino acid was purchased from Gibco (Gibco, Grand Island, NY, USA); 2-mercaptoethanol was obtained from AMRESCO (AMRESCO, solon OH, USA); fetal bovine serum (FBS) was obtained from Hyclone (Thermo scientific, IL, USA); antibodies against IKKβ, phospho-p38, p38, phospho-JNK, JNK, phospho-ERK1/2, ERK1/2, p65, phospho-p65, PLCγ1, phospho-PLCγ1 and SRC-3 were purchased from Cell Signaling Technology (Danvers, MA, USA); antibody against IκBα and Syk was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibody against phospho-Syk was purchased from ProSci (ProSci INCORPORATED, CA, USA).

Techniques:

Journal: Cell & Bioscience

Article Title: Deficiency in steroid receptor coactivator 3 enhances cytokine production in IgE-stimulated mast cells and passive systemic anaphylaxis in mice

doi: 10.1186/2045-3701-4-21

Figure Lengend Snippet: Passive cutaneous anaphylaxis in SRC-3 +/+ and SRC-3 -/- mice. SRC-3 +/+ (n = 6) and SRC-3 -/- mice (n = 6) were sensitized with anti-DNP IgE and DNP-HSA to induce cutaneous anaphylaxis as described in methods (A-D) . A , dye extravasation was observed after DNP-HSA injection at the injection sites in the ears. Photographs of the mice were taken 90 min after DNP-HSA administration. Representative images are shown. B , Extravasation of Evan’s blue was quantified as described in methods. Values are expressed as means + SD from three independent experiments. C , Toludine blue staining of mast cells in the ear skin of SRC-3 +/+ and SRC-3 -/- mice after antigen challenge. Representative images are shown; arrows indicate degranulated tissue mast cells. D , mast cells were quantified, values are expressed as means + SD from three independent experiments.

Article Snippet: RPMI 1640, DMSO, sodium pyruvate, antibody against β-actin and flag, anti-DNP IgE (clone SPE-7) were obtained from Sigma Aldrich (Sigma, St Louis, MO, USA); DNP-human serum albumin (DNP-HSA) was purchased from Biosearch Technologies (Biosearch Technologies, Novato CA, USA); recombinant murine SCF and IL-3 were purchased from peprotech (Peprotech, Rocky Hill, NJ, USA); anti-mouse CD117 (c-Kit)-PE, anti-mouse FcϵRI-FITC, IL-6 ELISA kit, and TNF-α ELISA kit were obtained from eBioscence (eBioscence, San Diego, CA, USA); nonessential amino acid was purchased from Gibco (Gibco, Grand Island, NY, USA); 2-mercaptoethanol was obtained from AMRESCO (AMRESCO, solon OH, USA); fetal bovine serum (FBS) was obtained from Hyclone (Thermo scientific, IL, USA); antibodies against IKKβ, phospho-p38, p38, phospho-JNK, JNK, phospho-ERK1/2, ERK1/2, p65, phospho-p65, PLCγ1, phospho-PLCγ1 and SRC-3 were purchased from Cell Signaling Technology (Danvers, MA, USA); antibody against IκBα and Syk was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibody against phospho-Syk was purchased from ProSci (ProSci INCORPORATED, CA, USA).

Techniques: Injection, Staining

Journal: Cell & Bioscience

Article Title: Deficiency in steroid receptor coactivator 3 enhances cytokine production in IgE-stimulated mast cells and passive systemic anaphylaxis in mice

doi: 10.1186/2045-3701-4-21

Figure Lengend Snippet: The degranulation of SRC-3 +/+ and SRC-3 -/- BMMCs. (A) Identification of BMMCs. Bone marrow cells were obtained from BALB/c mice and cultured in BMMC-complete medium. After 5 weeks, cells were identified by flow cytometric analysis for FcϵRI and c-kit expression. The experiment was repeated for 3 ~ 5 times. Representative results are shown from three independent experiments. (B) BMMCs were stimulated with IgE and different concentration of DNP-HSA. Degranulation was measured by assessing hexosaminidase activity in the media or cell lysates. Values are shown as the mean + SD from three independent experiments.

Article Snippet: RPMI 1640, DMSO, sodium pyruvate, antibody against β-actin and flag, anti-DNP IgE (clone SPE-7) were obtained from Sigma Aldrich (Sigma, St Louis, MO, USA); DNP-human serum albumin (DNP-HSA) was purchased from Biosearch Technologies (Biosearch Technologies, Novato CA, USA); recombinant murine SCF and IL-3 were purchased from peprotech (Peprotech, Rocky Hill, NJ, USA); anti-mouse CD117 (c-Kit)-PE, anti-mouse FcϵRI-FITC, IL-6 ELISA kit, and TNF-α ELISA kit were obtained from eBioscence (eBioscence, San Diego, CA, USA); nonessential amino acid was purchased from Gibco (Gibco, Grand Island, NY, USA); 2-mercaptoethanol was obtained from AMRESCO (AMRESCO, solon OH, USA); fetal bovine serum (FBS) was obtained from Hyclone (Thermo scientific, IL, USA); antibodies against IKKβ, phospho-p38, p38, phospho-JNK, JNK, phospho-ERK1/2, ERK1/2, p65, phospho-p65, PLCγ1, phospho-PLCγ1 and SRC-3 were purchased from Cell Signaling Technology (Danvers, MA, USA); antibody against IκBα and Syk was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibody against phospho-Syk was purchased from ProSci (ProSci INCORPORATED, CA, USA).

Techniques: Cell Culture, Expressing, Concentration Assay, Activity Assay

Journal: Cell & Bioscience

Article Title: Deficiency in steroid receptor coactivator 3 enhances cytokine production in IgE-stimulated mast cells and passive systemic anaphylaxis in mice

doi: 10.1186/2045-3701-4-21

Figure Lengend Snippet: Increased antigen-stimulated IL-6 and TNF-α expression from SRC-3 -/- BMMCs compared with SRC-3 +/+ BMMCs. BMMCs were pretreated with 1 μg/ml anti-DNP IgE for 4 h and then were stimulated with different concentration of DNP-HSA overnight at 37°C in 5% CO 2 . (A and B) The amounts of IL-6 and TNF-α in the medium were measured using ELISA assay kits. C and D , The mRNA levels of IL-6 and TNF-α were measured by real-time PCR. Values are shown as the mean + SD from three independent experiments. * p < 0.05.

Article Snippet: RPMI 1640, DMSO, sodium pyruvate, antibody against β-actin and flag, anti-DNP IgE (clone SPE-7) were obtained from Sigma Aldrich (Sigma, St Louis, MO, USA); DNP-human serum albumin (DNP-HSA) was purchased from Biosearch Technologies (Biosearch Technologies, Novato CA, USA); recombinant murine SCF and IL-3 were purchased from peprotech (Peprotech, Rocky Hill, NJ, USA); anti-mouse CD117 (c-Kit)-PE, anti-mouse FcϵRI-FITC, IL-6 ELISA kit, and TNF-α ELISA kit were obtained from eBioscence (eBioscence, San Diego, CA, USA); nonessential amino acid was purchased from Gibco (Gibco, Grand Island, NY, USA); 2-mercaptoethanol was obtained from AMRESCO (AMRESCO, solon OH, USA); fetal bovine serum (FBS) was obtained from Hyclone (Thermo scientific, IL, USA); antibodies against IKKβ, phospho-p38, p38, phospho-JNK, JNK, phospho-ERK1/2, ERK1/2, p65, phospho-p65, PLCγ1, phospho-PLCγ1 and SRC-3 were purchased from Cell Signaling Technology (Danvers, MA, USA); antibody against IκBα and Syk was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibody against phospho-Syk was purchased from ProSci (ProSci INCORPORATED, CA, USA).

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Cell & Bioscience

Article Title: Deficiency in steroid receptor coactivator 3 enhances cytokine production in IgE-stimulated mast cells and passive systemic anaphylaxis in mice

doi: 10.1186/2045-3701-4-21

Figure Lengend Snippet: SRC-3 deficiency has no effect on activation of Syk and PLCγ. BMMCs were stimulated with anti-DNP IgE 1 μg/ml for 4 h, and then treated with DNP-HSA 10 ng/ml for various times. Whole cell lysates were analyzed by western-blotting for Syk, p-Syk, PLCγ and p-PLCγ. Representative images are shown from three independent experiments.

Article Snippet: RPMI 1640, DMSO, sodium pyruvate, antibody against β-actin and flag, anti-DNP IgE (clone SPE-7) were obtained from Sigma Aldrich (Sigma, St Louis, MO, USA); DNP-human serum albumin (DNP-HSA) was purchased from Biosearch Technologies (Biosearch Technologies, Novato CA, USA); recombinant murine SCF and IL-3 were purchased from peprotech (Peprotech, Rocky Hill, NJ, USA); anti-mouse CD117 (c-Kit)-PE, anti-mouse FcϵRI-FITC, IL-6 ELISA kit, and TNF-α ELISA kit were obtained from eBioscence (eBioscence, San Diego, CA, USA); nonessential amino acid was purchased from Gibco (Gibco, Grand Island, NY, USA); 2-mercaptoethanol was obtained from AMRESCO (AMRESCO, solon OH, USA); fetal bovine serum (FBS) was obtained from Hyclone (Thermo scientific, IL, USA); antibodies against IKKβ, phospho-p38, p38, phospho-JNK, JNK, phospho-ERK1/2, ERK1/2, p65, phospho-p65, PLCγ1, phospho-PLCγ1 and SRC-3 were purchased from Cell Signaling Technology (Danvers, MA, USA); antibody against IκBα and Syk was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibody against phospho-Syk was purchased from ProSci (ProSci INCORPORATED, CA, USA).

Techniques: Activation Assay, Western Blot

Journal: Cell & Bioscience

Article Title: Deficiency in steroid receptor coactivator 3 enhances cytokine production in IgE-stimulated mast cells and passive systemic anaphylaxis in mice

doi: 10.1186/2045-3701-4-21

Figure Lengend Snippet: SRC-3 deficiency leads to increased activation of IKK-IκB-NF-κB pathway. BMMCs were stimulated with anti-DNP IgE 1 μg/ml for 4 h, and then treated with DNP-HSA 10 ng/ml for various times. Whole cell lysates were analyzed by western-blotting for IKK, p-IKK, IκB, p-IκB, p65 and p-p65. Representative images are shown from three independent experiments.

Article Snippet: RPMI 1640, DMSO, sodium pyruvate, antibody against β-actin and flag, anti-DNP IgE (clone SPE-7) were obtained from Sigma Aldrich (Sigma, St Louis, MO, USA); DNP-human serum albumin (DNP-HSA) was purchased from Biosearch Technologies (Biosearch Technologies, Novato CA, USA); recombinant murine SCF and IL-3 were purchased from peprotech (Peprotech, Rocky Hill, NJ, USA); anti-mouse CD117 (c-Kit)-PE, anti-mouse FcϵRI-FITC, IL-6 ELISA kit, and TNF-α ELISA kit were obtained from eBioscence (eBioscence, San Diego, CA, USA); nonessential amino acid was purchased from Gibco (Gibco, Grand Island, NY, USA); 2-mercaptoethanol was obtained from AMRESCO (AMRESCO, solon OH, USA); fetal bovine serum (FBS) was obtained from Hyclone (Thermo scientific, IL, USA); antibodies against IKKβ, phospho-p38, p38, phospho-JNK, JNK, phospho-ERK1/2, ERK1/2, p65, phospho-p65, PLCγ1, phospho-PLCγ1 and SRC-3 were purchased from Cell Signaling Technology (Danvers, MA, USA); antibody against IκBα and Syk was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibody against phospho-Syk was purchased from ProSci (ProSci INCORPORATED, CA, USA).

Techniques: Activation Assay, Western Blot

Journal: Cell & Bioscience

Article Title: Deficiency in steroid receptor coactivator 3 enhances cytokine production in IgE-stimulated mast cells and passive systemic anaphylaxis in mice

doi: 10.1186/2045-3701-4-21

Figure Lengend Snippet: SRC-3 deficiency leads to increased MAPK pathway activation. BMMCs were stimulated with anti-DNP IgE 1 μg/ml for 4 h, and then treated with DNP-HSA 10 ng/ml for various times. Whole cell lysates were analyzed by western-blotting for ERK, p-ERK, JNK, p-JNK, p38 and p-p38. Representative images are shown from three independent experiments.

Article Snippet: RPMI 1640, DMSO, sodium pyruvate, antibody against β-actin and flag, anti-DNP IgE (clone SPE-7) were obtained from Sigma Aldrich (Sigma, St Louis, MO, USA); DNP-human serum albumin (DNP-HSA) was purchased from Biosearch Technologies (Biosearch Technologies, Novato CA, USA); recombinant murine SCF and IL-3 were purchased from peprotech (Peprotech, Rocky Hill, NJ, USA); anti-mouse CD117 (c-Kit)-PE, anti-mouse FcϵRI-FITC, IL-6 ELISA kit, and TNF-α ELISA kit were obtained from eBioscence (eBioscence, San Diego, CA, USA); nonessential amino acid was purchased from Gibco (Gibco, Grand Island, NY, USA); 2-mercaptoethanol was obtained from AMRESCO (AMRESCO, solon OH, USA); fetal bovine serum (FBS) was obtained from Hyclone (Thermo scientific, IL, USA); antibodies against IKKβ, phospho-p38, p38, phospho-JNK, JNK, phospho-ERK1/2, ERK1/2, p65, phospho-p65, PLCγ1, phospho-PLCγ1 and SRC-3 were purchased from Cell Signaling Technology (Danvers, MA, USA); antibody against IκBα and Syk was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibody against phospho-Syk was purchased from ProSci (ProSci INCORPORATED, CA, USA).

Techniques: Activation Assay, Western Blot

Journal: Cell reports

Article Title: Hypoxic activation of PFKFB4 in breast tumor microenvironment shapes metabolic and cellular plasticity to accentuate metastatic competence

doi: 10.1016/j.celrep.2022.111756

Figure Lengend Snippet: (A and B) Representative PFKFB4 immunohistochemical staining of breast cancer patients (n = 122) with high or low staining of PFKFB4 in the nucleus (A) and cytoplasm (B). Scale bar, 300 μm. (C and D) Kaplan-Meier analysis of the overall survival of breast cancer patients (n = 122) with high or low expression of PFKFB4 in the nucleus (C) or cytoplasm (D). (E) WT and PFKFB4-KO MDA-MB-468 cells cultured in normoxic (21% O 2 ) or hypoxic condition (1% O 2 ) were subjected to subcellular fractionation, followed by immunoblot analyses of PFKFB4, β-tubulin, and lamin A/C. PFKFB4 blot was imaged for 30 s to obtain a low exposure and 120 s to obtain a high exposure. (F) Immunoblot analyses of nuclear fractions from MDA-MB-468 WT and PFKFB4-KO cells cultured in normoxic or hypoxic conditions were probed with PFKFB4, lamin A/C, and HIF-1α antibodies. (G) MDA-MB-468 cells cultured in normoxic or hypoxic conditions were used for immunofluorescence staining of PFKFB4. Scale bar, 25 μm (low-magnification image) and 10 μm (high-magnification image). (H and I) The mRNA expression of SLC2A1 (H) and PDK1 (I) in WT and PFKFB4-KO MDA-MB-468 cells under normoxic (21% O 2 ) or hypoxic condition (1% O 2 ) was measured by quantitative PCR (n = 3). Error bars are presented as mean ± SD. *p < 0.05; ***p < 0.001; ****p < 0.0001 by one-way ANOVA with Sidak’s multiple comparisons test. (J) HIF-1α luciferase (HRE) reporter assay in MDA-MB-468 WT and PFKFB4-KO cells expressing shNT or shSRC-3 cultured in normoxic (21% O 2 ) or hypoxic condition (1% O 2 ) for 24 h. Error bars are presented as mean ± SD. ****p < 0.0001; ns, not significant, calculated by one-way ANOVA with Sidak’s multiple comparisons test. (K) Immunoblot analyses of SRC-3 and integrin β3 in MDA-MB-231 cells stably expressing shNT or shSRC-3. β-actin was used as a loading control. See also and and .

Article Snippet: Rabbit monoclonal anti-phospho-SRC-3 (Ser857) (Clone 10A6) , Cell Signaling Technology (Dasgupta et al., 2018) , Cell culture supernatant produced from hybridoma; Gift from Cell Signaling Technology.

Techniques: Immunohistochemical staining, Staining, Expressing, Cell Culture, Fractionation, Western Blot, Immunofluorescence, Real-time Polymerase Chain Reaction, Luciferase, Reporter Assay, Stable Transfection

Journal: Cell reports

Article Title: Hypoxic activation of PFKFB4 in breast tumor microenvironment shapes metabolic and cellular plasticity to accentuate metastatic competence

doi: 10.1016/j.celrep.2022.111756

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-phospho-SRC-3 (Ser857) (Clone 10A6) , Cell Signaling Technology (Dasgupta et al., 2018) , Cell culture supernatant produced from hybridoma; Gift from Cell Signaling Technology.

Techniques: Cell Culture, Produced, Recombinant, Infection, Western Blot, Lysis, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Plasmid Preparation, Blocking Assay, Stripping, Magnetic Beads, SYBR Green Assay, Luciferase, Amplification, Sequencing, shRNA, Software, Real-time Polymerase Chain Reaction

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Endothelial Microparticle-Derived Reactive Oxygen Species: Role in Endothelial Signaling and Vascular Function

doi: 10.1155/2016/5047954

Figure Lengend Snippet: List of antibodies used.

Article Snippet: Total Src , Rabbit , Cell Signaling Technology , 2108 , 1 : 2000.

Techniques:

Journal: PLoS ONE

Article Title: Identification of Verrucarin A as a Potent and Selective Steroid Receptor Coactivator-3 Small Molecule Inhibitor

doi: 10.1371/journal.pone.0095243

Figure Lengend Snippet: (A) Chemical structure of verrucarin A. (B) Verrucarin A inhibits pBIND-SRC luciferase activity. HeLa cells were transiently cotransfected with expression vectors for pBIND-SRC-1, pBIND-SRC-2 or pBIND-SRC-3 and the GAL4-responsive pGL5 reporter plasmid before incubation with verrucarin A at different concentrations (0, 1, 2, 5, and 10 nM) for 24 h, followed by luciferase assay. Empty pBIND vector was transfected as a negative control. (C) Verrucarin A inhibits SRC coactivation of ERα. Luciferase assays were performed in HeLa cells transiently transfected with an ERE-luc reporter vector and expression vectors for ERα, and pCR3.1-SRC before incubation with 10 nM E2 and verrucarin A at different concentrations (0, 2, 5, and 10 nM) for 24 h.

Article Snippet: SRC-1 and SRC-3 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) and CARM1 and SRC-2 antibodies were purchased from Bethyl Laboratories (Montgomery, TX, USA). β-actin and p300 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Luciferase, Activity Assay, Expressing, Plasmid Preparation, Incubation, Transfection, Negative Control

Journal: PLoS ONE

Article Title: Identification of Verrucarin A as a Potent and Selective Steroid Receptor Coactivator-3 Small Molecule Inhibitor

doi: 10.1371/journal.pone.0095243

Figure Lengend Snippet: (A-B) A549 cells were treated with verrucarin A at different concentrations (0, 10, 20, 50, 100, and 200 nM) for 24 h, then Western analysis was performed to quantitate SRC-1, SRC-2, SRC-3, CARM1, and p300 proteins.

Article Snippet: SRC-1 and SRC-3 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) and CARM1 and SRC-2 antibodies were purchased from Bethyl Laboratories (Montgomery, TX, USA). β-actin and p300 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Identification of Verrucarin A as a Potent and Selective Steroid Receptor Coactivator-3 Small Molecule Inhibitor

doi: 10.1371/journal.pone.0095243

Figure Lengend Snippet: (A) Verrucarin A can kill a variety of cancer cells. MCF-7, A549, H1299, and PC-3 cells were treated with verrucarin A at different concentrations (0, 0.2, 0.5, 1, 2, 5, 10, and 20 nM) for 72 h, followed by MTS assay. (B) HepG2 cells are sensitive to verrucarin A, but primary hepatocytes are not. HepG2 cells were treated with verrucarin A at different concentrations (0, 0.2, 0.5, 1, 2, 5, 10, and 20 nM) for 48 h. Primary hepatocytes were treated with verrucarin A at different concentrations (0, 2, 5, 10, 20, 50, 100, and 200 nM) for 48 h, followed by MTS assay. (C) Verrucarin A inhibits cancer cell viabilities with potencies in line with its ability to down regulate SRC-3 protein levels.

Article Snippet: SRC-1 and SRC-3 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) and CARM1 and SRC-2 antibodies were purchased from Bethyl Laboratories (Montgomery, TX, USA). β-actin and p300 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: MTS Assay

Journal: PLoS ONE

Article Title: Identification of Verrucarin A as a Potent and Selective Steroid Receptor Coactivator-3 Small Molecule Inhibitor

doi: 10.1371/journal.pone.0095243

Figure Lengend Snippet: (A–B) Luciferase assays were performed in HeLa cells transiently transfected with MMP2-Luc, MMP13-Luc, and pCR3.1-SRC-3 expression vectors before incubation with verrucarin A at different concentrations (0, 2, 5, and 10 nM) for 24 h. (C) H1299 cells were treated with verrucarin A at different concentrations (0, 10, and 20 nM) for 24 h, then real-time PCR was performed to quantitate SRC-1, SRC-2, SRC-3, MMP2, and MMP13 mRNA expression.

Article Snippet: SRC-1 and SRC-3 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) and CARM1 and SRC-2 antibodies were purchased from Bethyl Laboratories (Montgomery, TX, USA). β-actin and p300 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Luciferase, Transfection, Expressing, Incubation, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Macrophage-Activating Lipopeptide-2 Requires Mal and PI3K for Efficient Induction of Heme Oxygenase-1

doi: 10.1371/journal.pone.0103433

Figure Lengend Snippet: A. THP-1 cells were stimulated with MALP-2 (5 ng/ml) for the indicated time points and lysed. The lysates were analyzed by Western blotting with a phosphorylated (p)-c-Src antibody (upper panel) or total c-Src antibody (lower panel). The results of a representative experiment are shown, and densitometric quantification of relative protein levels of p-c-Src from three experiments are presented under the panels. B. Cells were preincubated with PP1 for 1 h prior to stimulation with MALP-2. Cell lysates were prepared and HO-1 proteins were detected by Western blotting. C. Cells were transfected with c-Src specific siRNA or control (con) siRNA prior to 5.0 ng/ml MALP-2 treatment. Total c-Src and HO-1 protein expression levels were detected by Western blotting. Results shown are representative of at least three separate experiments. **, P<0.01 for significant difference between compared groups.

Article Snippet: Anti-HO-1, anti-phosphorylated and anti-total c-Src, anti- phosphorylated Btk, anti-phosphorylated Akt and anti-total Akt antibodies were products of Cell Signaling Technology (Beverly, MA).

Techniques: Western Blot, Transfection, Expressing

Journal: International Journal of Biological Sciences

Article Title: SRC-3 deficiency prevents atherosclerosis development by decreasing endothelial ICAM-1 expression to attenuate macrophage recruitment

doi: 10.7150/ijbs.74864

Figure Lengend Snippet: SRC-3 +/+ ApoE -/- mice exhibit more severe atherosclerosis. (A) SRC-3 was highly expressed in human atherosclerotic plaques. N represents plaque-adjacent vasculature in the lower limb aorta. AS represents atherosclerotic plaques in the lower limb aorta. (B) SRC-3 expression was upregulated in the aortas of ApoE -/- mice after the mice were fed a WD for 12 weeks. (C) SRC-3 was expressed in the endothelial cells and vascular smooth muscle cells of chow-fed SRC-3 -/- ApoE -/- mice and was further increased after the mice were fed a WD for 12 weeks. Sections of frozen aortic roots from SRC-3 +/+ ApoE -/- and SRC-3 -/- ApoE -/- mice were subjected to X-gal staining. Arrows indicate positively stained cells (blue). Scale bar, 100 µm. (D) SRC-3 promoted atherosclerotic plaque formation. Representative images of en face Oil Red O-stained aortas from SRC-3 +/+ ApoE -/- and SRC-3 -/- ApoE -/- mice (left panel). Quantification of the plaque areas in aortas (right panel). (E-K) Cross-sections of the aortic roots from SRC-3 +/+ ApoE -/- and SRC-3 -/- ApoE -/- mice were subjected to (E-F) H&E staining (scale bar, 500 µm (E); scale bar, 200 µm (F)), (G) α-SMA staining (scale bar, 200 µm), (H-I) Masson staining (scale bar, 200 µm (H); scale bar, 200 µm (I)), (J) Oil Red O staining (scale bar, 500 µm), and (K) F4/80 staining (scale bar, 200 µm). Left panels, representative images. Right panels, quantification of stained area or a percentage of lesion area. “×” indicates necrotic area. (L) Plaque stability was significantly increased in SRC-3 -/- ApoE -/- mice. The data represent the mean ± SEM. The results are representative of three independent experiments. P values were calculated by unpaired two-tailed Student's t-test. *, P <0.05; ** P <0.01.

Article Snippet: Anti-SRC-3 antibodies (C-20, sc-7216) and anti-p65 (D14E12, #8284) antibodies were purchased from Santa Cruz Biotechnology and Cell Signaling Technology, respectively.

Techniques: Expressing, Staining, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: SRC-3 deficiency prevents atherosclerosis development by decreasing endothelial ICAM-1 expression to attenuate macrophage recruitment

doi: 10.7150/ijbs.74864

Figure Lengend Snippet: SRC-3 in endothelial cells contributes to the development of atherosclerosis. (A) Western blot showing that AAV-mediated SRC-3 shRNA decreased SRC-3 expression levels in the aortas of ApoE -/- mice. (B) SRC-3 knockdown reduced WD-induced atherosclerotic plaque formation in ApoE -/- mice. Representative images of en face Oil Red O-stained aortas from ApoE -/- mice injected with AAV-mediated SRC-3 shRNA and scramble shRNA (left panel). Quantification of the plaque areas of aortas (right panel). The data represent the mean ± SEM. (C-F) Cross-sections of the aortic roots of ApoE -/- mice injected with AAV-mediated SRC-3 shRNA and scramble shRNA were subjected to (C) H&E staining (scale bar, 100 µm), (D) Masson staining (scale bar, 500 µm), (E) Oil Red O staining (scale bar, 100 µm), (F) F4/80 staining (scale bar, 100 µm). Left panels, representative images. Right panels, quantification of stained area or a percentage of lesion area. The data represent the mean ± SEM. The results are representative of three independent experiments. P values were calculated by unpaired two-tailed Student's t-test. *, P <0.05; **, P <0.01; ***, P <0.001.

Article Snippet: Anti-SRC-3 antibodies (C-20, sc-7216) and anti-p65 (D14E12, #8284) antibodies were purchased from Santa Cruz Biotechnology and Cell Signaling Technology, respectively.

Techniques: Western Blot, shRNA, Expressing, Staining, Injection, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: SRC-3 deficiency prevents atherosclerosis development by decreasing endothelial ICAM-1 expression to attenuate macrophage recruitment

doi: 10.7150/ijbs.74864

Figure Lengend Snippet: SRC-3 increases ICAM-1 expression during atherosclerosis development. (A) KEGG enrichment pathway analysis and (B) Gene Ontology (GO) biological process analysis of mRNA profiles in the aortas of SRC-3 +/+ ApoE -/- and SRC-3 -/- ApoE -/- mice after WD feeding for 12 weeks. (C) Selected genes involved in leukocyte recruitment and proinflammatory markers are shown as a heat map. (D) The mRNA level of ICAM-1 in the aortas of SRC-3 -/- ApoE -/- mice was significantly decreased after WD feeding for 12 weeks. (E) The protein level of ICAM-1 in the aortas of SRC-3 +/+ ApoE -/- and SRC-3 -/- ApoE -/- mice after WD feeding for 12 weeks. Each lane represents a pooled sample of three representative mice. (F) Western blot analysis of SRC-3 and ICAM-1 in 16 atherosclerotic plaques and plaque-adjacent vasculature in the lower limb aorta of accident patients. N represents plaque-adjacent vasculature in the lower limb aorta; AS represents atherosclerotic plaques in the lower limb aorta. (G) Correlation between SRC-3 and ICAM-1 protein levels in 16 atherosclerotic plaques and plaque-adjacent vasculature in the lower limb aorta. The data represent the mean ± SEM. The results are representative of three independent experiments. P values were calculated by unpaired two-tailed Student's t-test. *, P <0.05.

Article Snippet: Anti-SRC-3 antibodies (C-20, sc-7216) and anti-p65 (D14E12, #8284) antibodies were purchased from Santa Cruz Biotechnology and Cell Signaling Technology, respectively.

Techniques: Expressing, Western Blot, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: SRC-3 deficiency prevents atherosclerosis development by decreasing endothelial ICAM-1 expression to attenuate macrophage recruitment

doi: 10.7150/ijbs.74864

Figure Lengend Snippet: SRC-3 regulates ICAM-1 expression via enhancing NF-κB signaling. (A) The protein levels of SRC-3 and ICAM-1 in SRC-3 siRNA-transfected HUVECs were significantly reduced compared with those in scrambled siRNA-transfected HUVECs after TNFα or IL-1β treatment. (B) The mRNA level of ICAM-1 in the SRC-3 siRNA-transfected HUVECs was markedly decreased after TNFα or IL-1β treatment. (C) ICAM-1 promoter activity was reduced in SRC-3 siRNA-transfected HUVECs after TNFα or IL-1β treatment. (D) SRC-3 cooperated with p65 to enhance the activity of the NF-κB reporter (upper panel) and ICAM-1 promoter (lower panel). (E) NF-κB binding site mutation abolished NF-κB-mediated ICAM-1 promoter activity. (F) The recruitment of SRC-3 and p65 was significantly reduced in SRC-3 siRNA-transfected HUVECs after TNFα or IL-1β treatment (right panel). Position of the subfragments detected by ChIP assays (left panel). (G) The SRC-3 siRNA-transfected HUVECs monolayer exhibited a significantly decreased number of adhered (upper panel) and transmigrated (lower panel) THP-1 cells after TNFα or IL-1β treatment. (H) Western blot showing ICAM-1 overexpression in SRC-3 siRNA-transfected HUVECs. (I) ICAM-1 overexpression in SRC-3 siRNA-transfected HUVECs rescued monocyte attachment to HUVECs and monocyte transendothelial migration after TNFα or IL-1β treatment. The data represent the mean ± SEM of three independent experiments. P values were calculated by unpaired two-tailed Student's t-test. *, P <0.05; **, P <0.01;***, P <0.001.

Article Snippet: Anti-SRC-3 antibodies (C-20, sc-7216) and anti-p65 (D14E12, #8284) antibodies were purchased from Santa Cruz Biotechnology and Cell Signaling Technology, respectively.

Techniques: Expressing, Transfection, Activity Assay, Binding Assay, Mutagenesis, Western Blot, Over Expression, Migration, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: SRC-3 deficiency prevents atherosclerosis development by decreasing endothelial ICAM-1 expression to attenuate macrophage recruitment

doi: 10.7150/ijbs.74864

Figure Lengend Snippet: Pharmacological inhibition of SRC-3 reduces atherosclerosis. (A) The protein levels of SRC-3, ICAM-1 and p-p65 in bufalin-treated HUVECs were significantly reduced compared with those in vehicle-treated HUVECs after TNFα or IL-1β treatment. The bufalin-treated HUVECs monolayer resulted in a dramatically decreased number of adhered (upper panel) and transmigrated (lower panel) THP-1 cells after TNFα or IL-1β treatment. (B-C) ApoE -/- mice were administered vehicle or bufalin (1.0 mg/kg, six times a week) by intraperitoneal injection for 13 weeks concomitant with WD feeding. (B) Dosing regimen (ApoE -/- prevention model). (C) Representative images of en face Oil Red O-stained aortas from ApoE -/- mice treated with vehicle or bufalin (left panel). Quantification of the plaque areas of aortas (right panel). The data represent the mean ± SEM. (D) The protein levels of SRC-3 and ICAM-1 in the aortas of ApoE -/- mice treated with bufalin were significantly reduced in the ApoE -/- prevention model. (E-F) ApoE -/- mice were fed a WD for 10 weeks and then treated with vehicle or bufalin (1.0 mg/kg, six times a week) by intraperitoneal injection for 13 weeks concomitant with WD feeding. (E) Dosing regimen (ApoE -/- regression model). (F) Representative images of en face Oil Red O-stained aortas from ApoE -/- mice treated with vehicle or bufalin (left panel). Quantification of the plaque areas of aortas (right panel). The data represent the mean ± SEM. (G) The protein levels of SRC-3 and ICAM-1 in the aortas of ApoE -/- mice treated with bufalin were significantly reduced in the ApoE -/- regression model. The data represent the mean ± SEM. The results are representative of three independent experiments. P values were calculated by unpaired two-tailed Student's t-test. *, P <0.05; **, P <0.01;***, P <0.001.

Article Snippet: Anti-SRC-3 antibodies (C-20, sc-7216) and anti-p65 (D14E12, #8284) antibodies were purchased from Santa Cruz Biotechnology and Cell Signaling Technology, respectively.

Techniques: Inhibition, Injection, Staining, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: SRC-3 deficiency prevents atherosclerosis development by decreasing endothelial ICAM-1 expression to attenuate macrophage recruitment

doi: 10.7150/ijbs.74864

Figure Lengend Snippet: Schematic model of the mechanism by which SRC-3 accelerates atherosclerosis development. SRC-3 promotes atherosclerosis development by increasing ICAM-1 transcription by enhancing the function of NF-κB in endothelial cells to promote macrophage recruitment. SRC-3 depletion or pharmacological inhibition of SRC-3 by bufalin ameliorates atherosclerosis development through decreasing endothelial ICAM-1 expression and macrophage recruitment via reduction of NF-κB function.

Article Snippet: Anti-SRC-3 antibodies (C-20, sc-7216) and anti-p65 (D14E12, #8284) antibodies were purchased from Santa Cruz Biotechnology and Cell Signaling Technology, respectively.

Techniques: Inhibition, Expressing

Journal: International Journal of Biological Sciences

Article Title: SRC-3 deficiency prevents atherosclerosis development by decreasing endothelial ICAM-1 expression to attenuate macrophage recruitment

doi: 10.7150/ijbs.74864

Figure Lengend Snippet: SRC-3 +/+ ApoE -/- mice exhibit more severe atherosclerosis. (A) SRC-3 was highly expressed in human atherosclerotic plaques. N represents plaque-adjacent vasculature in the lower limb aorta. AS represents atherosclerotic plaques in the lower limb aorta. (B) SRC-3 expression was upregulated in the aortas of ApoE -/- mice after the mice were fed a WD for 12 weeks. (C) SRC-3 was expressed in the endothelial cells and vascular smooth muscle cells of chow-fed SRC-3 -/- ApoE -/- mice and was further increased after the mice were fed a WD for 12 weeks. Sections of frozen aortic roots from SRC-3 +/+ ApoE -/- and SRC-3 -/- ApoE -/- mice were subjected to X-gal staining. Arrows indicate positively stained cells (blue). Scale bar, 100 µm. (D) SRC-3 promoted atherosclerotic plaque formation. Representative images of en face Oil Red O-stained aortas from SRC-3 +/+ ApoE -/- and SRC-3 -/- ApoE -/- mice (left panel). Quantification of the plaque areas in aortas (right panel). (E-K) Cross-sections of the aortic roots from SRC-3 +/+ ApoE -/- and SRC-3 -/- ApoE -/- mice were subjected to (E-F) H&E staining (scale bar, 500 µm (E); scale bar, 200 µm (F)), (G) α-SMA staining (scale bar, 200 µm), (H-I) Masson staining (scale bar, 200 µm (H); scale bar, 200 µm (I)), (J) Oil Red O staining (scale bar, 500 µm), and (K) F4/80 staining (scale bar, 200 µm). Left panels, representative images. Right panels, quantification of stained area or a percentage of lesion area. “×” indicates necrotic area. (L) Plaque stability was significantly increased in SRC-3 -/- ApoE -/- mice. The data represent the mean ± SEM. The results are representative of three independent experiments. P values were calculated by unpaired two-tailed Student's t-test. *, P <0.05; ** P <0.01.

Article Snippet: Equal amounts of proteins were loaded onto 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred onto polyvinylidene difluoride membranes (Millipore), followed by immunoblotting with anti-SRC-3 (Cell Signaling Technology, 5E11, #2116), anti-human ICAM-1 (Abcam, EPR4776, ab109361), anti-mouse ICAM-1 (Abcam, EPR16608, ab179707), anti-p65 (Cell Signaling Technology, D14E12, #8284), anti-p-p65 (Cell Signaling Technology, Ser536, #3031), anti-GAPDH (Cell Signaling Technology, D16H11, #5174) and anti-β-actin (Sigma, AC-15, #A5441).

Techniques: Expressing, Staining, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: SRC-3 deficiency prevents atherosclerosis development by decreasing endothelial ICAM-1 expression to attenuate macrophage recruitment

doi: 10.7150/ijbs.74864

Figure Lengend Snippet: SRC-3 in endothelial cells contributes to the development of atherosclerosis. (A) Western blot showing that AAV-mediated SRC-3 shRNA decreased SRC-3 expression levels in the aortas of ApoE -/- mice. (B) SRC-3 knockdown reduced WD-induced atherosclerotic plaque formation in ApoE -/- mice. Representative images of en face Oil Red O-stained aortas from ApoE -/- mice injected with AAV-mediated SRC-3 shRNA and scramble shRNA (left panel). Quantification of the plaque areas of aortas (right panel). The data represent the mean ± SEM. (C-F) Cross-sections of the aortic roots of ApoE -/- mice injected with AAV-mediated SRC-3 shRNA and scramble shRNA were subjected to (C) H&E staining (scale bar, 100 µm), (D) Masson staining (scale bar, 500 µm), (E) Oil Red O staining (scale bar, 100 µm), (F) F4/80 staining (scale bar, 100 µm). Left panels, representative images. Right panels, quantification of stained area or a percentage of lesion area. The data represent the mean ± SEM. The results are representative of three independent experiments. P values were calculated by unpaired two-tailed Student's t-test. *, P <0.05; **, P <0.01; ***, P <0.001.

Article Snippet: Equal amounts of proteins were loaded onto 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred onto polyvinylidene difluoride membranes (Millipore), followed by immunoblotting with anti-SRC-3 (Cell Signaling Technology, 5E11, #2116), anti-human ICAM-1 (Abcam, EPR4776, ab109361), anti-mouse ICAM-1 (Abcam, EPR16608, ab179707), anti-p65 (Cell Signaling Technology, D14E12, #8284), anti-p-p65 (Cell Signaling Technology, Ser536, #3031), anti-GAPDH (Cell Signaling Technology, D16H11, #5174) and anti-β-actin (Sigma, AC-15, #A5441).

Techniques: Western Blot, shRNA, Expressing, Staining, Injection, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: SRC-3 deficiency prevents atherosclerosis development by decreasing endothelial ICAM-1 expression to attenuate macrophage recruitment

doi: 10.7150/ijbs.74864

Figure Lengend Snippet: SRC-3 increases ICAM-1 expression during atherosclerosis development. (A) KEGG enrichment pathway analysis and (B) Gene Ontology (GO) biological process analysis of mRNA profiles in the aortas of SRC-3 +/+ ApoE -/- and SRC-3 -/- ApoE -/- mice after WD feeding for 12 weeks. (C) Selected genes involved in leukocyte recruitment and proinflammatory markers are shown as a heat map. (D) The mRNA level of ICAM-1 in the aortas of SRC-3 -/- ApoE -/- mice was significantly decreased after WD feeding for 12 weeks. (E) The protein level of ICAM-1 in the aortas of SRC-3 +/+ ApoE -/- and SRC-3 -/- ApoE -/- mice after WD feeding for 12 weeks. Each lane represents a pooled sample of three representative mice. (F) Western blot analysis of SRC-3 and ICAM-1 in 16 atherosclerotic plaques and plaque-adjacent vasculature in the lower limb aorta of accident patients. N represents plaque-adjacent vasculature in the lower limb aorta; AS represents atherosclerotic plaques in the lower limb aorta. (G) Correlation between SRC-3 and ICAM-1 protein levels in 16 atherosclerotic plaques and plaque-adjacent vasculature in the lower limb aorta. The data represent the mean ± SEM. The results are representative of three independent experiments. P values were calculated by unpaired two-tailed Student's t-test. *, P <0.05.

Article Snippet: Equal amounts of proteins were loaded onto 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred onto polyvinylidene difluoride membranes (Millipore), followed by immunoblotting with anti-SRC-3 (Cell Signaling Technology, 5E11, #2116), anti-human ICAM-1 (Abcam, EPR4776, ab109361), anti-mouse ICAM-1 (Abcam, EPR16608, ab179707), anti-p65 (Cell Signaling Technology, D14E12, #8284), anti-p-p65 (Cell Signaling Technology, Ser536, #3031), anti-GAPDH (Cell Signaling Technology, D16H11, #5174) and anti-β-actin (Sigma, AC-15, #A5441).

Techniques: Expressing, Western Blot, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: SRC-3 deficiency prevents atherosclerosis development by decreasing endothelial ICAM-1 expression to attenuate macrophage recruitment

doi: 10.7150/ijbs.74864

Figure Lengend Snippet: SRC-3 regulates ICAM-1 expression via enhancing NF-κB signaling. (A) The protein levels of SRC-3 and ICAM-1 in SRC-3 siRNA-transfected HUVECs were significantly reduced compared with those in scrambled siRNA-transfected HUVECs after TNFα or IL-1β treatment. (B) The mRNA level of ICAM-1 in the SRC-3 siRNA-transfected HUVECs was markedly decreased after TNFα or IL-1β treatment. (C) ICAM-1 promoter activity was reduced in SRC-3 siRNA-transfected HUVECs after TNFα or IL-1β treatment. (D) SRC-3 cooperated with p65 to enhance the activity of the NF-κB reporter (upper panel) and ICAM-1 promoter (lower panel). (E) NF-κB binding site mutation abolished NF-κB-mediated ICAM-1 promoter activity. (F) The recruitment of SRC-3 and p65 was significantly reduced in SRC-3 siRNA-transfected HUVECs after TNFα or IL-1β treatment (right panel). Position of the subfragments detected by ChIP assays (left panel). (G) The SRC-3 siRNA-transfected HUVECs monolayer exhibited a significantly decreased number of adhered (upper panel) and transmigrated (lower panel) THP-1 cells after TNFα or IL-1β treatment. (H) Western blot showing ICAM-1 overexpression in SRC-3 siRNA-transfected HUVECs. (I) ICAM-1 overexpression in SRC-3 siRNA-transfected HUVECs rescued monocyte attachment to HUVECs and monocyte transendothelial migration after TNFα or IL-1β treatment. The data represent the mean ± SEM of three independent experiments. P values were calculated by unpaired two-tailed Student's t-test. *, P <0.05; **, P <0.01;***, P <0.001.

Article Snippet: Equal amounts of proteins were loaded onto 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred onto polyvinylidene difluoride membranes (Millipore), followed by immunoblotting with anti-SRC-3 (Cell Signaling Technology, 5E11, #2116), anti-human ICAM-1 (Abcam, EPR4776, ab109361), anti-mouse ICAM-1 (Abcam, EPR16608, ab179707), anti-p65 (Cell Signaling Technology, D14E12, #8284), anti-p-p65 (Cell Signaling Technology, Ser536, #3031), anti-GAPDH (Cell Signaling Technology, D16H11, #5174) and anti-β-actin (Sigma, AC-15, #A5441).

Techniques: Expressing, Transfection, Activity Assay, Binding Assay, Mutagenesis, Western Blot, Over Expression, Migration, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: SRC-3 deficiency prevents atherosclerosis development by decreasing endothelial ICAM-1 expression to attenuate macrophage recruitment

doi: 10.7150/ijbs.74864

Figure Lengend Snippet: Pharmacological inhibition of SRC-3 reduces atherosclerosis. (A) The protein levels of SRC-3, ICAM-1 and p-p65 in bufalin-treated HUVECs were significantly reduced compared with those in vehicle-treated HUVECs after TNFα or IL-1β treatment. The bufalin-treated HUVECs monolayer resulted in a dramatically decreased number of adhered (upper panel) and transmigrated (lower panel) THP-1 cells after TNFα or IL-1β treatment. (B-C) ApoE -/- mice were administered vehicle or bufalin (1.0 mg/kg, six times a week) by intraperitoneal injection for 13 weeks concomitant with WD feeding. (B) Dosing regimen (ApoE -/- prevention model). (C) Representative images of en face Oil Red O-stained aortas from ApoE -/- mice treated with vehicle or bufalin (left panel). Quantification of the plaque areas of aortas (right panel). The data represent the mean ± SEM. (D) The protein levels of SRC-3 and ICAM-1 in the aortas of ApoE -/- mice treated with bufalin were significantly reduced in the ApoE -/- prevention model. (E-F) ApoE -/- mice were fed a WD for 10 weeks and then treated with vehicle or bufalin (1.0 mg/kg, six times a week) by intraperitoneal injection for 13 weeks concomitant with WD feeding. (E) Dosing regimen (ApoE -/- regression model). (F) Representative images of en face Oil Red O-stained aortas from ApoE -/- mice treated with vehicle or bufalin (left panel). Quantification of the plaque areas of aortas (right panel). The data represent the mean ± SEM. (G) The protein levels of SRC-3 and ICAM-1 in the aortas of ApoE -/- mice treated with bufalin were significantly reduced in the ApoE -/- regression model. The data represent the mean ± SEM. The results are representative of three independent experiments. P values were calculated by unpaired two-tailed Student's t-test. *, P <0.05; **, P <0.01;***, P <0.001.

Article Snippet: Equal amounts of proteins were loaded onto 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred onto polyvinylidene difluoride membranes (Millipore), followed by immunoblotting with anti-SRC-3 (Cell Signaling Technology, 5E11, #2116), anti-human ICAM-1 (Abcam, EPR4776, ab109361), anti-mouse ICAM-1 (Abcam, EPR16608, ab179707), anti-p65 (Cell Signaling Technology, D14E12, #8284), anti-p-p65 (Cell Signaling Technology, Ser536, #3031), anti-GAPDH (Cell Signaling Technology, D16H11, #5174) and anti-β-actin (Sigma, AC-15, #A5441).

Techniques: Inhibition, Injection, Staining, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: SRC-3 deficiency prevents atherosclerosis development by decreasing endothelial ICAM-1 expression to attenuate macrophage recruitment

doi: 10.7150/ijbs.74864

Figure Lengend Snippet: Schematic model of the mechanism by which SRC-3 accelerates atherosclerosis development. SRC-3 promotes atherosclerosis development by increasing ICAM-1 transcription by enhancing the function of NF-κB in endothelial cells to promote macrophage recruitment. SRC-3 depletion or pharmacological inhibition of SRC-3 by bufalin ameliorates atherosclerosis development through decreasing endothelial ICAM-1 expression and macrophage recruitment via reduction of NF-κB function.

Article Snippet: Equal amounts of proteins were loaded onto 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred onto polyvinylidene difluoride membranes (Millipore), followed by immunoblotting with anti-SRC-3 (Cell Signaling Technology, 5E11, #2116), anti-human ICAM-1 (Abcam, EPR4776, ab109361), anti-mouse ICAM-1 (Abcam, EPR16608, ab179707), anti-p65 (Cell Signaling Technology, D14E12, #8284), anti-p-p65 (Cell Signaling Technology, Ser536, #3031), anti-GAPDH (Cell Signaling Technology, D16H11, #5174) and anti-β-actin (Sigma, AC-15, #A5441).

Techniques: Inhibition, Expressing

Journal: International Journal of Biological Sciences

Article Title: SRC-3 protects intestine from DSS-induced colitis by inhibiting inflammation and promoting goblet cell differentiation through enhancement of KLF4 expression

doi: 10.7150/ijbs.28576

Figure Lengend Snippet: SRC-3 -/- mice are more susceptible to DSS-induced colitis compared with wild-type mice. (A) Survival of SRC-3 -/- mice and wild-type mice after oral administration of 2% DSS dissolved in sterile distill water for 7 days. Survival curve was calculated by the log-rank methods. Results were calculated from three independent experiments. Body weight change (B), combined scores of stool consistency (C) and bleeding scores (D) of SRC-3 -/- mice (n = 13) and wild-type mice (n = 15) after oral administration of 2% DSS dissolved in sterile distill water for 7 days. Macroscopic pictures (E) and colonic length (F) of SRC-3 -/- mice (n = 8) and wild-type mice (n = 8) after oral administration of 2% DSS dissolved in sterile distill water for 7 days. Pictures are representative of three independent experiments. * p <0.05, ** p <0.01.

Article Snippet: Antigens were retrieved by citrate buffer (pH 6.0) under microwave heating for 15 min. To block nonspecific binding sites, sections were incubated with 10% goat serum for 10 min at room temperature and then were incubated with anti-SRC-3 (cell signaling, 5E11, #2116) and anti-MUC2 (Proteintech Group, 27675-1-AP) for overnight at 4℃.

Techniques:

Journal: International Journal of Biological Sciences

Article Title: SRC-3 protects intestine from DSS-induced colitis by inhibiting inflammation and promoting goblet cell differentiation through enhancement of KLF4 expression

doi: 10.7150/ijbs.28576

Figure Lengend Snippet: SRC-3 -/- mice display more severe histopathology and produce more proinflammatory cytokines than do wild-type mice after DSS administration. (A) H&E staining of colon from SRC-3 -/- mice and wild-type mice without or with DSS administration (n = 6-8). (B) Histopathological scoring of crypt damage and inflammation in the colon from SRC-3 -/- mice and wild-type mice without or with DSS administration (n = 6-8). Arrow and arrowhead denote significant submucosal inflammatory cell infiltration and crypt damage. (C) The concentrations of IL-1β, IL-6, TNF-α, IFN-γ, and CCL2 in the colons of SRC-3 -/- mice and wild-type mice without or with DSS administration (n = 6-8). Results are representative of three independent experiments. * p <0.05, ** p <0.01.

Article Snippet: Antigens were retrieved by citrate buffer (pH 6.0) under microwave heating for 15 min. To block nonspecific binding sites, sections were incubated with 10% goat serum for 10 min at room temperature and then were incubated with anti-SRC-3 (cell signaling, 5E11, #2116) and anti-MUC2 (Proteintech Group, 27675-1-AP) for overnight at 4℃.

Techniques: Histopathology, Staining

Journal: International Journal of Biological Sciences

Article Title: SRC-3 protects intestine from DSS-induced colitis by inhibiting inflammation and promoting goblet cell differentiation through enhancement of KLF4 expression

doi: 10.7150/ijbs.28576

Figure Lengend Snippet: SRC-3 -/- mice suffer a severe loss of mature colonic goblet cells. (A) Representative AB/PAS staining of the colon sections from SRC-3 -/- mice (n = 6) and wild-type mice (n = 10) at days 0, 4, and 6 post-DSS administration (left panels). Quantification of AB/PAS positive cells (right panels). Arrow and arrowhead denote AB-positive cells (blue) and PAS-positive cells (pink), respectively. (B) Representative MUC2 immunostaining of the colon sectins from SRC-3 -/- mcie (n = 5) and wild-type mice (n = 5) at days 0, 4 and 6 post-DSS administration (left panels). Quantification of MUC2-positive cells (right panels). Arrow denotes MUC2-positive cells (brown). (C) Representative AB/PAS staining of the colon sections from SRC-3 -/- mice and wild-type mice (left panels). Arrow represents inner mucus layer. Quantification of thickness of inner mucus layer (right panels). Data are mean + SEM. * p <0.05, ** p <0.01.

Article Snippet: Antigens were retrieved by citrate buffer (pH 6.0) under microwave heating for 15 min. To block nonspecific binding sites, sections were incubated with 10% goat serum for 10 min at room temperature and then were incubated with anti-SRC-3 (cell signaling, 5E11, #2116) and anti-MUC2 (Proteintech Group, 27675-1-AP) for overnight at 4℃.

Techniques: Staining, Immunostaining

Journal: International Journal of Biological Sciences

Article Title: SRC-3 protects intestine from DSS-induced colitis by inhibiting inflammation and promoting goblet cell differentiation through enhancement of KLF4 expression

doi: 10.7150/ijbs.28576

Figure Lengend Snippet: Transcription factor KLF4 is decreased in the colons of SRC-3 -/- mice compared to wild-type mice. Quantitative RT-PCR of GFI1 (A) , SPDEF (B) , HES5 (C) , and KLF4 (D) in the colons of SRC-3 -/- mice (n = 6) and wild-type mice (n = 10) at days 0, 4, and 6 post-DSS administration. (E) The protein levels of KLF4 were detected by western blot in the colonic epithelial cells of SRC-3 -/- mice and wild-type mice at day 0 and 6 post-DSS administration. Data are mean + SEM. Results are representative of three independent experiments. * p <0.05, ** p <0.01.

Article Snippet: Antigens were retrieved by citrate buffer (pH 6.0) under microwave heating for 15 min. To block nonspecific binding sites, sections were incubated with 10% goat serum for 10 min at room temperature and then were incubated with anti-SRC-3 (cell signaling, 5E11, #2116) and anti-MUC2 (Proteintech Group, 27675-1-AP) for overnight at 4℃.

Techniques: Quantitative RT-PCR, Western Blot

Journal: International Journal of Biological Sciences

Article Title: SRC-3 protects intestine from DSS-induced colitis by inhibiting inflammation and promoting goblet cell differentiation through enhancement of KLF4 expression

doi: 10.7150/ijbs.28576

Figure Lengend Snippet: SRC-3 promotes KLF4 expression through cooperating with transcription factor c-Fos. (A) KLF4 mRNA expression was reduced in two SRC-3-knockdown stable LS174T cell lines (shSRC-3-1 and shSRC-3-2). Data are mean + SD (n = 3). (B) The expression of KLF4 and MUC2 proteins was reduced in two SRC-3-knockdown LS174T cell lines. (C) SRC-3 knockdown inhibited goblet cell differentiation of LS174T cells. Goblet cell differentiation was assessed by PAS staining. (D) KLF4 overexpression partly rescued MUC2 expression in SRC-3-knockdown LS174T cells. (E) KLF4 overexpression rescued SRC-3-knockdown-induced losses of PAS staining. (F) KLF4 promoter activity was decreased in shSRC-3-1 and shSRC-3-2 LS174T cells. Data are mean + SD (n = 3). (G) SRC-3 cooperated with c-Fos to enhance the activity of the KLF4 promoter. (H) Mutation analysis of the role of c-Fos binding site in c-Fos/SRC-3-mediated activation of the KLF4 promoter. LS174T cells were transfected with wild-type KLF4 promoter reporter (pKLF4-Fos-WT) and c-Fos binding site mutated KLF4 promoter reporter (pKLF4-Fos-Mut) together with SRC-3 expression plasmid and c-Fos expression plasmid, respectively. KLF4 promoter activity was assayed at 24 h post-transfection. Data are mean + SD (n = 3). (I) KLF4 protein was reduced in c-Fos-knockdown LS174T cells. (J) KLF4 mRNA expression was decreased in c-Fos-knockdown LS174T cells. Data are mean + SD (n = 3). (K) KLF4 promoter activity was decreased in c-Fos-knockdown LS174T cells. Data are mean + SD (n = 3). * p <0.05, ** p <0.01.

Article Snippet: Antigens were retrieved by citrate buffer (pH 6.0) under microwave heating for 15 min. To block nonspecific binding sites, sections were incubated with 10% goat serum for 10 min at room temperature and then were incubated with anti-SRC-3 (cell signaling, 5E11, #2116) and anti-MUC2 (Proteintech Group, 27675-1-AP) for overnight at 4℃.

Techniques: Expressing, Cell Differentiation, Staining, Over Expression, Activity Assay, Mutagenesis, Binding Assay, Activation Assay, Transfection, Plasmid Preparation

Journal: International Journal of Biological Sciences

Article Title: SRC-3 protects intestine from DSS-induced colitis by inhibiting inflammation and promoting goblet cell differentiation through enhancement of KLF4 expression

doi: 10.7150/ijbs.28576

Figure Lengend Snippet: Both c-Fos and SRC-3 are recruited to c-Fos binding site at the KLF4 promoter. (A) c-Fos could be recruited to KLF4 promoter, and the recruitment was increased after c-Fos overexpression. (B) SRC-3 could also be recruited to KLF4 promoter, and the recruitment was increased after c-Fos overexpression. (C) SRC-3 knockdown reduced SRC-3 recruitment to KLF4 promoter. (K) SRC-3 knockdown reduced c-Fos recruitment to KLF4 promoter. Data are mean +SD (n = 3). *p<0.05, *** p <0.001.

Article Snippet: Antigens were retrieved by citrate buffer (pH 6.0) under microwave heating for 15 min. To block nonspecific binding sites, sections were incubated with 10% goat serum for 10 min at room temperature and then were incubated with anti-SRC-3 (cell signaling, 5E11, #2116) and anti-MUC2 (Proteintech Group, 27675-1-AP) for overnight at 4℃.

Techniques: Binding Assay, Over Expression

Journal: International Journal of Biological Sciences

Article Title: SRC-3 protects intestine from DSS-induced colitis by inhibiting inflammation and promoting goblet cell differentiation through enhancement of KLF4 expression

doi: 10.7150/ijbs.28576

Figure Lengend Snippet: SRC-3 interacts with c-Fos through the S/T and HAT domains of SRC-3. (A) Co-IP analysis of the interaction of SRC-3 and c-Fos in 293T cells. (B) Co-IP analysis of the interaction of SRC-3 and c-Fos in LS174T cells. (C) SRC-3 interacted with c-Fos through its S/T and HAT domains.

Article Snippet: Antigens were retrieved by citrate buffer (pH 6.0) under microwave heating for 15 min. To block nonspecific binding sites, sections were incubated with 10% goat serum for 10 min at room temperature and then were incubated with anti-SRC-3 (cell signaling, 5E11, #2116) and anti-MUC2 (Proteintech Group, 27675-1-AP) for overnight at 4℃.

Techniques: Co-Immunoprecipitation Assay