Journal: Stem cell research & therapy

Article Title: Intracellular osteopontin potentiates the immunosuppressive activity of mesenchymal stromal cells.

doi: 10.1186/s13287-024-03979-8

Figure Lengend Snippet: Fig. 5 iOPN promoted STAT1-mediated immunosuppression in MSCs. a–d Vector-MSCs and iOPN-MSCs or WT-MSCs and OPN−/−-MSCs were treated with or without TNF-α plus IFN-γ (10 ng/mL) for the indicated times. Cells were harvested, and OPN, NF-κB p65, STAT1, IKBα, phosphorylation of NF-κB p65, phosphorylation of STAT1 at Tyr701 and phosphorylation of IKBα were analyzed by immunoblotting analysis. Full-length blots are presented in Additional file 1: Fig. 5a-d. e–g Vector-MSCs and iOPN-MSCs were stimulated with TNF-α plus IFN-γ (10 ng/mL) for 12 h. The STAT1 inhibitor fludarabine (Flu, 2 μM) was then added to the culture medium of Vector-MSCs and iOPN-MSCs. The expression of iNOS was determined by quantitative real-time PCR (e), immunoblotting analysis (f) and flow cytometry (g). Full-length blots are presented in Additional file 1: Fig. 5f. h Vector-MSCs and iOPN-MSCs were pretreated with DMSO or fludarabine (Flu, 2 μM) for 6 h and then irradiated and cocultured with CFSE-labeled splenocytes activated by anti-CD3/CD28 antibodies for 3 days at a ratio of 1:20. CD4+ T cells and CD8+ T cells were stained for proliferation analysis by flow cytometry at the end of coculture, and the percentages of proliferating T cells are shown. The results are representative of three to six independent experiments. Values are shown as the mean ± SEM and statistical significance is indicated as *P < 0.05, **P < 0.01 and ***P < 0.001

Article Snippet: Antibodies against GAPDH (CAT# 2118, RRID: AB_561053), NF-κB p65 (CAT# 8242, RRID:AB_10859369), phospho-NF-κB p65 (CAT# 3033, RRID:AB_331284), STAT1 (CAT# 14994, RRID:AB_2737027), phospho-STAT1 (Tyr701) (CAT# 7649, RRID:AB_10950970), IκBα (CAT# 4814, RRID:AB_390781), phospho-IκBα (CAT# 2118) and ubiquitin (CAT# 3936, RRID: RRID:AB_331292) were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA).

Techniques: Plasmid Preparation, Phospho-proteomics, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Irradiation, Labeling, Staining

Journal: Oncology Letters

Article Title: Sorafenib induced alteration of protein glycosylation in hepatocellular carcinoma cells

doi: 10.3892/ol.2017.6177

Figure Lengend Snippet: Results of lectin blotting and western blotting. (A) Lectin blotting validated Sorafenib induced alteration by the biotinylated MAL-I and biotinylated PHA-L. Anti-β-actin antibody was used to show the protein loading. (B) Downregulation of p-ERK in sorafenib-treated MHCC97L and MHCC97H cells. (C) Downregulation of p-ERK in MHCC97L and MHCC97H cells with U0126 treatment. (D) Diagram of the possible mechanism by which sorafenib treatment reduced the expression of Ets-1. sorafenib treatment decreased the expression of Ets-1 by blocking the Ras/Raf/MAPK signaling pathway in HCC cells. MAL-1, Maackia amurensis lecin I; PHA-L, Phaseolus vulgaris leucoagglutinin; p-, phosphorylated; MEK, mitogen activated protein kinase; ERK, extracellular signal-related kinases; Ets-1, erythroblastosis-26; U0126, 1,4-Diamino-2,3-dicyano-1,4-bis (2-aminophenylthio) butadiene.

Article Snippet: The antibodies used are as follows: Polyclonal rabbit anti-human Ets-1 antibody (dilution, 1:1,000; cat. no. 6258; Cell Signaling Technology, Danvers, MA, USA); monoclonal rabbit anti-human extracellular signal-related kinase (ERK) antibody (dilution, 1:2,000; cat. no. 4695; Cell Signaling Technology); monoclonal rabbit anti-human phospho-ERK antibody (p-ERK; dilution, 1:2,000; cat. no. 4370; Cell Signaling Technology); and a monoclonal mouse anti-human β-actin antibody (dilution, 1:2,000).

Techniques: Western Blot, Expressing, Blocking Assay

Journal: Molecular medicine reports

Article Title: Survivin activates NF‑κB p65 via the IKKβ promoter in esophageal squamous cell carcinoma.

doi: 10.3892/mmr.2015.4737

Figure Lengend Snippet: Figure 1. Survivin knockdown results in downregulation of IKKα, IΚΚβ and NF‑κB p65 in Eca109 and KYSE150 cells. Eca109 and KYSE150 cells were transfected with LV3‑survivin shRNA plasmid (Eca109 survivin KD and KYSE150 survivin KD) and LV3‑survivin control plasmids (Eca109 control and KYSE150 control), respectively. The expression levels of survivin, NF‑κB, IΚΚα and IΚΚβ were measured by quantitative reverse transcrip tion‑polymerase chain reaction and western blotting in (A and B) Eca109 cells and (C and D) KYSE150 cells. GADPH served as an internal and loading control. Columns present the mean values from triplicate experiments and the error bars indicate the standard deviation. *P<0.05; **P<0.01 vs. control. KD, knockdown; NF‑κB p65, transcription factor p65; IKKα, inhibitor of nuclear factor κB kinase subunit α; IΚΚβ, inhibitor of nuclear factor κB kinase subunit β; p, phosphorylated.

Article Snippet: The membranes were blocked with 5% non-fat dried milk (Sigma-Aldrich) in Tris-buffered saline and Tween-20 (TBST; Beijing Solarbio Science & Technology Co., Ltd.,Beijing,China) at room temperature for 2 h. The membranes were incubated overnight at 4 ̊C with the following primary antibodies: Rabbit polyclonal anti-GAPDH (1:400; cat. no. BA2913; Wuhan Boster Biological Technology, Ltd.), which served as a loading control; rabbit polyclonal anti-survivin (1:1,000; cat. no. sc-10811; Santa Cruz Biotechnology Inc.); rabbit polyclonal anti-phosphorylated (p)-NF-κB p65 (pSer536) (1:1,000; cat. no. AF2006; Affinity Biosciences, Cell Signal Transduction, Cincinnati, OH, USA); rabbit polyclonal anti-NF-κB p65 (1:400; cat. no. BA0610; Wuhan Boster Biological Technology, Ltd.); rabbit polyclonal anti‐IΚΚα (1:400; cat. no. BA1594-2; Wuhan Boster Biological Technology, Ltd.); and rabbit polyclonal anti‐IΚΚβ (1:400; cat. no. BA4458-2; Wuhan Boster Biological Technology, Ltd.) were blocked in TBST.

Techniques: Knockdown, Transfection, shRNA, Plasmid Preparation, Control, Expressing, Western Blot, Standard Deviation

Journal: Molecular medicine reports

Article Title: Survivin activates NF‑κB p65 via the IKKβ promoter in esophageal squamous cell carcinoma.

doi: 10.3892/mmr.2015.4737

Figure Lengend Snippet: Figure 2. YM155 inhibited the expression of survivin, NF‑κB p65, IKKα and IΚΚβ in Eca109 and KYSE150 cells. (A) Eca109 and KYSE150 cells were incubated with 0.005, 0.05, 0.5, 5 and 50 µM YM155 for 48 h. Cells incubated with the culture medium served as the control group. Experiments were performed in triplicate. Eca109 and KYSE150 cells were treated with 0 (blank), 0.05, 0.5, and 5.0 µM YM155. The expression of survivin, NF‑κB p65, IΚΚβ and IΚΚα were analyzed by (B and C) western blotting and (D and E) RT‑qPCR. Western blotting of survivin, p‑NF‑κB p65, NF‑κB p65, IΚΚβ and IΚΚα proteins was conducted using total protein isolated from cells, with GADPH serving as a loading control. Transcript levels were measured by RT‑qPCR of total isolated RNA, with GADPH serving as an internal control. YM155 inhibited expression of survivin and NF‑κB p65, IKKα and IΚΚβ in (B and D) Eca109 and (C and E) KYSE150 cells. Columns demonstrate the mean values from triplicate experiments and the error bars indicate standard deviation. *P<0.05; **P<0.01 vs. control. RT‑qPCR, quantitative reverse transcription‑polymerase chain reaction; NF‑κB p65, transcription factor p65; IKKα, inhibitor of nuclear factor κB kinase subunit α; IΚΚβ, inhibitor of nuclear factor κB kinase subunit β; p, phosphorylated; mRNA, messenger RNA.

Article Snippet: The membranes were blocked with 5% non-fat dried milk (Sigma-Aldrich) in Tris-buffered saline and Tween-20 (TBST; Beijing Solarbio Science & Technology Co., Ltd.,Beijing,China) at room temperature for 2 h. The membranes were incubated overnight at 4 ̊C with the following primary antibodies: Rabbit polyclonal anti-GAPDH (1:400; cat. no. BA2913; Wuhan Boster Biological Technology, Ltd.), which served as a loading control; rabbit polyclonal anti-survivin (1:1,000; cat. no. sc-10811; Santa Cruz Biotechnology Inc.); rabbit polyclonal anti-phosphorylated (p)-NF-κB p65 (pSer536) (1:1,000; cat. no. AF2006; Affinity Biosciences, Cell Signal Transduction, Cincinnati, OH, USA); rabbit polyclonal anti-NF-κB p65 (1:400; cat. no. BA0610; Wuhan Boster Biological Technology, Ltd.); rabbit polyclonal anti‐IΚΚα (1:400; cat. no. BA1594-2; Wuhan Boster Biological Technology, Ltd.); and rabbit polyclonal anti‐IΚΚβ (1:400; cat. no. BA4458-2; Wuhan Boster Biological Technology, Ltd.) were blocked in TBST.

Techniques: Expressing, Incubation, Control, Western Blot, Isolation, Standard Deviation, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular medicine reports

Article Title: Survivin activates NF‑κB p65 via the IKKβ promoter in esophageal squamous cell carcinoma.

doi: 10.3892/mmr.2015.4737

Figure Lengend Snippet: Figure 3. Survivin overexpression results in upregulation of NF‑κBp65, IKKα, IΚΚβ in Eca109 and KYSE150cells. (A and B) Eca109 and (C and D) KYSE150 cells were transfected with GV142‑survivin overexpression plasmid (Eca109 survivin OE and KYSE150 survivin OE), GV142‑control plasmid (Eca109 control and KYSE150 control), and mock transfection. Expression levels of survivin, NF‑κB p65, IΚΚβ, and IΚΚα were analyzed by (A and C) RT‑qPCR and (B and D) western blotting of total protein. Transcript levels were measured by RT‑qPCR of total isolated RNA, with GADPH serving as an internal control. Columns indicate the mean values from triplicate experiments and the error bars indicate standard deviation. *P<0.05; **P<0.01 vs. control. OE, overexpression; RT‑qPCR, quantitative reverse transcription‑polymerase chain reaction; NF‑κB p65, transcription factor p65; IKKα, inhibitor of nuclear factor κB kinase subunit α; IΚΚβ, inhibitor of nuclear factor κB kinase subunit β; p, phosphorylated; mRNA, messenger RNA.

Article Snippet: The membranes were blocked with 5% non-fat dried milk (Sigma-Aldrich) in Tris-buffered saline and Tween-20 (TBST; Beijing Solarbio Science & Technology Co., Ltd.,Beijing,China) at room temperature for 2 h. The membranes were incubated overnight at 4 ̊C with the following primary antibodies: Rabbit polyclonal anti-GAPDH (1:400; cat. no. BA2913; Wuhan Boster Biological Technology, Ltd.), which served as a loading control; rabbit polyclonal anti-survivin (1:1,000; cat. no. sc-10811; Santa Cruz Biotechnology Inc.); rabbit polyclonal anti-phosphorylated (p)-NF-κB p65 (pSer536) (1:1,000; cat. no. AF2006; Affinity Biosciences, Cell Signal Transduction, Cincinnati, OH, USA); rabbit polyclonal anti-NF-κB p65 (1:400; cat. no. BA0610; Wuhan Boster Biological Technology, Ltd.); rabbit polyclonal anti‐IΚΚα (1:400; cat. no. BA1594-2; Wuhan Boster Biological Technology, Ltd.); and rabbit polyclonal anti‐IΚΚβ (1:400; cat. no. BA4458-2; Wuhan Boster Biological Technology, Ltd.) were blocked in TBST.

Techniques: Over Expression, Transfection, Plasmid Preparation, Control, Expressing, Western Blot, Isolation, Standard Deviation, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular medicine reports

Article Title: HAT inhibitor, garcinol, exacerbates lipopolysaccharide‑induced inflammation in vitro and in vivo.

doi: 10.3892/mmr.2016.5189

Figure Lengend Snippet: Figure 2. GAR suppresses LPS‑induced acetylation of nuclear factor‑κB p65 in RAW264.7 macrophages. RAW264.7 cells were incubated with vehicle or 10 µmol/l GAR in the presence or absence of 100 µg/l LPS. The cells were harvested 18 h subsequent to LPS challenge. (A) Protein levels of acetylated p65 (Lys310) and total p65 were determined by western blot analysis. The bands of acetylated p65 and p65 are indicated by arrows. (B) Densitometry was performed and data are presented as relative intensity units. Data are expressed as the mean ± standard deviation, n=4. *P<0.05 vs. LPS group. GAR, garcinol; LPS, lipopolysaccharide.

Article Snippet: Monoclonal rabbit anti‐mouse p65 (cat. no. 8242) and monoclonal rabbit anti‐mouse acetyl‐p65 (Lys310; cat. no. 12629) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Incubation, Western Blot, Standard Deviation

Journal: Molecular Medicine Reports

Article Title: Isorhamnetin inhibits cell proliferation and induces apoptosis in breast cancer via Akt and mitogen-activated protein kinase kinase signaling pathways

doi: 10.3892/mmr.2015.4269

Figure Lengend Snippet: Effects of isorhamnetin on the cell signaling cascade. (A) Isorhamnetin inhibited the phosphorylation of Akt, mTOR, MEK1/2 and ERK1/2, but not of EGFR (12-h treatment). The protein expression levels were quantified by Image J, and presented as the relative expression levels to the control. (B) Isorhamnetin increased the expression level of Bax and cleaved caspase 3, and decreased the expression level of Bcl-2 and Bcl-xL (12-h treatment); (C) Isorhamnetin decreased the nuclear translocation of NF-κB (24-h treatment). p, phosphorylated; mTOR, mammalian target of rapamycin; MEK, mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinase; EGFR, epidermal growth factor receptor; PI3K, phosphoinositide 3-kinase; Bcl-2, B cell lymphoma 2; Bax, Bcl-2-associated protein X; Bcl-2-xL, Bcl-extra large; NF-κB, nuclear factor-κB. * P<0.05, vs. the control group.

Article Snippet: The antibodies for mouse monoclonal β-actin (cat. no. 3700; 1:1,000), rabbit polyclonal phosphorylated (p)-EGFR immunoglobulin (Ig)G (cat. no. 2234; 1:1,000), rabbit monoclonal EGFR (cat. no. 4405; 1:1,000), rabbit monoclonal PI3K (cat. no. 4249; 1:1,000), rabbit monoclonal p-Akt (S473; cat. no. 4060; 1:500), mouse monoclonal Akt (cat. no. 2920; 1:1,000), mouse monoclonal p-ERK1/2 (cat. no. 9106; 1:500), rabbit monoclonal ERK1/2 (cat. no. 4695; 1:1,000) and rabbit monoclonal cleaved caspase-3 (cat. no. 9664; 1:500) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA), and antibodies for rabbit monoclonal B cell lymphoma 2 (Bcl-2; cat. no. ab117115; 1:1,000), rabbit monoclonal Bcl-2-associated X protein (Bax; cat. no. ab32503; 1:1,000), rabbit monoclonal Bcl-extra large (xL; cat. no. ab32370; 1:2,000), rabbit monoclonal IκB (cat. no. ab32518; 1:1,000), rabbit monoclonal anti-NF-κB P65 antibody (cat. no. ab32536; 1:2,000) and rabbit polyclonal H3 (cat. no. ab1791; 1:2,000) were purchased from Abcam (Cambridge, MA, USA).

Techniques: Expressing, Translocation Assay

Journal: Molecular Medicine Reports

Article Title: Isorhamnetin inhibits cell proliferation and induces apoptosis in breast cancer via Akt and mitogen-activated protein kinase kinase signaling pathways

doi: 10.3892/mmr.2015.4269

Figure Lengend Snippet: JSH-23, the NF-κB inhibitor, did not contribute to the induction of apoptosis by isorhamnetin in the MCF7 cells. (A) Induction of caspase-3 cleavage was not increased by treatment with JSH-23 (12-h treatment); (B) Induction of cell apoptosis was not augmented by the NF-kB inhibitor, JSH-23 (48 h treatment). NF-κB, nuclear factor-κB; Con, control; PI, propidium iodide; JSH, JSH-23; Iso, isorhamnetin.

Article Snippet: The antibodies for mouse monoclonal β-actin (cat. no. 3700; 1:1,000), rabbit polyclonal phosphorylated (p)-EGFR immunoglobulin (Ig)G (cat. no. 2234; 1:1,000), rabbit monoclonal EGFR (cat. no. 4405; 1:1,000), rabbit monoclonal PI3K (cat. no. 4249; 1:1,000), rabbit monoclonal p-Akt (S473; cat. no. 4060; 1:500), mouse monoclonal Akt (cat. no. 2920; 1:1,000), mouse monoclonal p-ERK1/2 (cat. no. 9106; 1:500), rabbit monoclonal ERK1/2 (cat. no. 4695; 1:1,000) and rabbit monoclonal cleaved caspase-3 (cat. no. 9664; 1:500) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA), and antibodies for rabbit monoclonal B cell lymphoma 2 (Bcl-2; cat. no. ab117115; 1:1,000), rabbit monoclonal Bcl-2-associated X protein (Bax; cat. no. ab32503; 1:1,000), rabbit monoclonal Bcl-extra large (xL; cat. no. ab32370; 1:2,000), rabbit monoclonal IκB (cat. no. ab32518; 1:1,000), rabbit monoclonal anti-NF-κB P65 antibody (cat. no. ab32536; 1:2,000) and rabbit polyclonal H3 (cat. no. ab1791; 1:2,000) were purchased from Abcam (Cambridge, MA, USA).

Techniques:

Journal: BMC Musculoskeletal Disorders

Article Title: Expression and pathological effects of periostin in human osteoarthritis cartilage

doi: 10.1186/s12891-015-0682-3

Figure Lengend Snippet: Periostin-induced upregulation of catabolic enzymes and inflammatory cytokines via the NF-κB signaling. ( a ) Human primary chondrocytes were stimulated by 20 μg/mL periostin, and the nuclear translocation of p65 was determined by immunocytochemistry at 3 h. Gray and green represent p65. Red represent nucleus. Bar = 10 μm. ( b ) Human primary chondrocytes were exposed to 20 μg/mL periostin and/or different concentrations of BAY11-7082 for 24 h. The mRNA levels of IL-6, IL-8, MMP-1, MMP-3, MMP-13, and NOS2 were measured by quantitative RT-PCR. The relative expression compared to vehicle control is shown. Representative data from one of three donors are shown. *, P < 0.05

Article Snippet: The cells were incubated with rabbit anti-p65 antibody (1:100, Cat. sc109; Santa Cruz Biotechnology) for 1 h and then with Alexa Fluor 488-conjugated anti-rabbit secondary antibody (1:300, Thermo Scientific) for 1 h. Nuclei were stained with DAPI.

Techniques: Translocation Assay, Immunocytochemistry, Quantitative RT-PCR, Expressing, Control