Journal: Journal of Nanobiotechnology

Article Title: Dental pulp stem cell-derived exosomes suppress M1 macrophage polarization through the ROS-MAPK-NFκB P65 signaling pathway after spinal cord injury

doi: 10.1186/s12951-022-01273-4

Figure Lengend Snippet: DPI can suppress macrophages M1 polarization and MAPK-NFκB P65 signaling pathway. A and B Raw264.7 cells were treated with LPS, LPS + DPI or LPS + DPI + H 2 O 2 . M1 macrophages surface expression (CD86) was detected using using flow cytometry. The LPS + DPI group had a markedly lower M1 polarization rate than the LPS group. Furthermore, the LPS + DPI + H 2 O 2 group had a significantly increased M1 polarization rate compared with the LPS + DPI group. C and D The ERK1/2 and p-ERK1/2 protein expression in Raw264.7 cells was either investigated. LPS upregulated the P-ERK/ERK level in RAW264.7 cells and that DPI downregulated the LPS-induced increase in the P-ERK/ERK level. The LPS + DPI + H2O2 group had a higher P-ERK/ERK level than the LPS + DPI group. E–G P65 protein expression are determined by immunofluorescences and flow cytometry. LPS could increase P65 expression. DPI had a marked inhibitory effect on the increase in P65 fluorescence, and H2O2 counteracted the inhibitory effect of DPI.*p < 0.05, ***p < 0.01

Article Snippet: The cells were incubated with anti-p65-Alexa Fluor 488 (CST 494455) for 1 h at room temperature in the dark.

Techniques: Expressing, Flow Cytometry, Fluorescence

Journal: Journal of Nanobiotechnology

Article Title: Dental pulp stem cell-derived exosomes suppress M1 macrophage polarization through the ROS-MAPK-NFκB P65 signaling pathway after spinal cord injury

doi: 10.1186/s12951-022-01273-4

Figure Lengend Snippet: ROS activates macrophages M1 polarization through MAPK-NFκB P65 signaling pathway. A and B Raw264.7 cells were treated with H 2 O 2 or H 2 O 2 + DPI. M1 macrophages surface expression (CD86) was detected using flow cytometry. H2O2 could promote M1 macrophage polarization, but DPI had no effect on M1 macrophage polarization induced by H2O2. C and D The ERK1/2 and p-ERK1/2 protein expression in Raw264.7 cells was either investigated. H2O2 could increase the P-ERK/ERK level and that DPI had no influence on the phosphorylation of ERK in cells cultured with H2O2. E–G P65 protein expression are determined by immunofluorescences and flow cytometry. P65 expression was also increased after H2O2 treatment for 24 h, and DPI could not suppress the P65 fluorescence increase caused by H2O2. H and I Raw264.7 cells were treated with H 2 O 2 , H 2 O 2 + BAY 11–7082 or H 2 O 2 + PD98059. J and K Raw264.7 cells were treated with LPS, LPS + BAY 11–7082 or LPS + PD98059. L–N P65 protein expression are determined by immunofluorescences and flow cytometry. M1 macrophages surface expression (CD86) was detected using using flow cytometry. macrophage M1 polarization caused by both LPS and H2O2 could be suppressed by the P65 inhibitor. PD98059, an inhibitor of MEK, could also inhibit P65 expression and that M1 macrophage polarization was increased by LPS or H2O2. *p < 0.05, ***p < 0.01

Article Snippet: The cells were incubated with anti-p65-Alexa Fluor 488 (CST 494455) for 1 h at room temperature in the dark.

Techniques: Expressing, Flow Cytometry, Phospho-proteomics, Cell Culture, Fluorescence

Journal: Journal of Nanobiotechnology

Article Title: Dental pulp stem cell-derived exosomes suppress M1 macrophage polarization through the ROS-MAPK-NFκB P65 signaling pathway after spinal cord injury

doi: 10.1186/s12951-022-01273-4

Figure Lengend Snippet: Characteristics of DPSCs derived Exosomes. DPSC derived exosomes could inhibit macrophages M1 polarization through MAPK-NFκB P65 signaling pathway in vivo. A Representative images of exosomes are observed under transmission electron microscopy (TEM). Size distribution of extracellular vesicle is measured by nanoparticle tracking analysis (NTA, ZetaView, Particle Metrix Inc., German). Western-blotting analysis of indicated proteins is detected, including CD63, CD9, tubulin, albumin and the MSC marker CD73. B and C Raw264.7 cells were treated with LPS or LPS + EXO for 2, 4, 6 and 24 h. ROS level was detected by using FACS with a flow cytometry. The LPS + Exos group had a lower ROS level than the LPS group. D and E Raw264.7 cells were treated with LPS or LPS + EXO for 24 h. M1 macrophages surface expression (CD86) was detected using using flow cytometry. DPSC-derived exosomes could lower the LPS-induced increase in the M1 polarization rate. F and G The ERK1/2 and p-ERK1/2 protein expression in Raw264.7 cells was either investigated. LPS + Exos group had a lower P-ERK/ERK level than the LPS group. H–J P65 protein expression are determined by immunofluorescences and flow cytometry. DPSC-derived exosomes also inhibited the increase in P65 fluorescence induced by LPS. *p < 0.05, ***p < 0.01

Article Snippet: The cells were incubated with anti-p65-Alexa Fluor 488 (CST 494455) for 1 h at room temperature in the dark.

Techniques: Derivative Assay, In Vivo, Transmission Assay, Electron Microscopy, Western Blot, Marker, Flow Cytometry, Expressing, Fluorescence

Journal: Journal of Nanobiotechnology

Article Title: Dental pulp stem cell-derived exosomes suppress M1 macrophage polarization through the ROS-MAPK-NFκB P65 signaling pathway after spinal cord injury

doi: 10.1186/s12951-022-01273-4

Figure Lengend Snippet: DPSC derived exosomes can inhibit ROS-MAPK-NFκB P65 signaling pathway in vivo. ROS level was detected by using FACS with a flow cytometry. A and B Mice are analyzed at 3 days after injured spinal cord. C and D Mice are analyzed at 5 days after injured spinal cord. There was a marked increase in ROS levels in the injured spinal cord at 3 days and 5 days. E and F At 3 days, the ERK1/2 and p-ERK1/2 protein expression in spinal tissues was investigated. G and H At 5 days, the ERK1/2 and p-ERK1/2 protein expression in spinal tissues was either investigated. There was also a higher P-ERK/ERK level in the SCI group than in the sham group at 3 days and 5 days. *p < 0.05, ***p < 0.01.

Article Snippet: The cells were incubated with anti-p65-Alexa Fluor 488 (CST 494455) for 1 h at room temperature in the dark.

Techniques: Derivative Assay, In Vivo, Flow Cytometry, Expressing

Journal: Journal of Nanobiotechnology

Article Title: Dental pulp stem cell-derived exosomes suppress M1 macrophage polarization through the ROS-MAPK-NFκB P65 signaling pathway after spinal cord injury

doi: 10.1186/s12951-022-01273-4

Figure Lengend Snippet: DPSC derived exosomes can inhibit macrophages M1 polarization in mice. A At 3 days after injured spinal cord, P65 and CD86 were determined by immunofuorescence in different groups. B At 5 days after injured spinal cord, P65 and CD86 were determined by immunofluorescences in different groups. P65 and CD86 fluorescence intensity around the injury site was increased in the SCI group 3 days and 5 days after SCI

Article Snippet: The cells were incubated with anti-p65-Alexa Fluor 488 (CST 494455) for 1 h at room temperature in the dark.

Techniques: Derivative Assay, Fluorescence

Journal: Microbiology Spectrum

Article Title: Epstein-Barr Virus Envelope Glycoprotein gp110 Inhibits IKKi-Mediated Activation of NF-κB and Promotes the Degradation of β-Catenin

doi: 10.1128/spectrum.00326-23

Figure Lengend Snippet: gp110 disrupts IKKi-mediated activation of the NF-κB pathway. (A) HEK293T cells were transfected with gp110-HA expression plasmid or vector along with or without IKKi-Flag expression plasmid to activate the phosphorylation of p65 (Ser536). At 24 h posttransfection, the cell lysates were collected for WB with rabbit anti-p65 pAb, mouse anti-Flag MAb, and mouse anti-HA MAb. Rabbit anti-p65 (Ser536) pAb was used to detect the phosphorylation of p65, and β-actin was used as the loading control. (B and C) gp110-HA expression plasmid or vector was individually transfected into HEK293T cells (C) or cotransfected with the plasmid combination of IKKi-Myc/p65-Flag into HEK293T cells (B). At 24 h posttransfection, cells were infected with or without 100 HAU/mL SeV for 16 h, collected, and lysed. The samples were then used for Co-IP analysis with mouse anti-Myc MAb (B) or rabbit anti-p65 pAb (C). Immunoprecipitated proteins were then resolved by 10% SDS-PAGE, and WB was performed with mouse anti-Flag MAb, mouse anti-Myc MAb, and mouse anti-HA MAb. Rabbit anti-IKKi pAb and rabbit anti-p65 pAbs were used to detect the expression of endogenous IKKi and p65 (C), respectively, and β-actin was used as the loading control. (D) HEK293T cells were transfected with different concentrations of gp110-HA expression plasmid or vector. At 24 h posttransfection, cells were infected with or without 100 HAU/mL SeV for 16 h. Cell lysates were collected for WB with rabbit anti-p65 pAb and mouse anti-HA MAb. Rabbit anti-p65 (Ser536) pAb was used to detect the phosphorylation of p65, and β-actin was used as the loading control. (E) Hone1-EBV cells were transfected with the expression plasmid of shBALF4 or pSuper vector. At 24 h posttransfection, cells were treated with or without TPA (40 ng/mL) and NaB (3 mM) for 24 h to induce lytic EBV infection. Cells were then infected with or without 100 HAU/mL SeV for 16 h, and cell lysates were collected for WB with mouse anti-gp110 MAb, rabbit anti-p65 pAb, and rabbit anti-p65 (Ser536) pAb. β-Actin was used as the loading control. (F) HEK293T cells were transfected with gp110-HA expression plasmid or vector. At 24 h posttransfection, cells were infected with or without 100 HAU/mL SeV for 16 h. Cell lysates were harvested for cellular fractionation, and WB was performed with mouse anti-HA MAb and rabbit anti-p65 pAb. (G) Hone1-EBV cells were transfected with the expression plasmid of shBALF4 or pSuper vector. At 24 h posttransfection, cells were treated with or without TPA (40 ng/mL) and NaB (3 mM) for 24 h to induce lytic EBV infection. Cells were infected with or without 100 HAU/mL SeV for 16 h, and cell lysates were collected for WB with mouse anti-gp110 MAb and rabbit anti-p65 pAb. Here, GAPDH was used as the cytosol marker, and histone 3 was used as the nucleus marker. The gray analysis was calculated using ImageJ.

Article Snippet: Rabbit anti-ubiquitin MAb, anti-IRF3 (Ser396), and anti-p65 (Ser536) phosphorylated pAbs, alkaline phosphatase (AP)-labeled goat anti-mouse IgG, and goat anti-rabbit IgG were purchased from Cell Signaling Technology.

Techniques: Activation Assay, Transfection, Expressing, Plasmid Preparation, Phospho-proteomics, Control, Infection, Co-Immunoprecipitation Assay, Immunoprecipitation, SDS Page, Cell Fractionation, Marker

Journal: Oncogene

Article Title: The Prolyl Isomerase Pin1 Regulates the NF-κB Signaling Pathway and Interleukin-8 Expression in Glioblastoma

doi: 10.1038/onc.2009.232

Figure Lengend Snippet: (A) U251 shPin1 cells were grown in 150 mm dishes. When cell density reached approximately 80%, the cells were serum-starved for 4 h, treated with TNFα (10 ng/ml) for the indicated times (0-120 min), and lysed in RIPA buffer. Lysate from each sample was then subjected to immunoprecipitation (IP) using antibodies specific for Pin1 or normal rabbit serum (NRS). IP samples were probed with antibodies to p65 to demonstrate a specific interaction between p65 and Pin1. (B) U251.shPin1 cells were grown in 150 mm dishes in the absence or presence of Tet for 72 h. Cells were lysed in RIPA buffer and anti-p65 was used in the subsequent IP along with a normal mouse serum (NMS) control. Immunoblotting was performed using antibodies against Pin1, IκBα and p65. Results shown are representative of three experiments.

Article Snippet: Anti-p65 pS276 (3027) and anti-pIKKα/pIKKβ (2681) antibodies were from Cell Signaling (Danvers, MA).

Techniques: Immunoprecipitation, Control, Western Blot

Journal: Oncogene

Article Title: The Prolyl Isomerase Pin1 Regulates the NF-κB Signaling Pathway and Interleukin-8 Expression in Glioblastoma

doi: 10.1038/onc.2009.232

Figure Lengend Snippet: (A) U251 shPin1 cells were grown in the absence or presence of Tet for 72 h. 1 × 10 4 cells were plated in 8-well chamber slides and allowed to recover overnight. Cells were then serum starved, stimulated with 10 ng/ml of TNFα for 1 h, and assayed by immunofluorescence with the indicated antibodies. Dapi was used to stain nuclei (blue) and the indicated antibodies were used to analyze protein levels and localization (red). Multiple images were taken from each chamber; representative images for each condition from three experiments are shown. (B) 2.7 × 10 5 U251.shPin1 cells were plated in 100 mm dishes and grown in the absence or presence of Tet. After 72 h, media was changed to serum-free and cells were serum-starved overnight. The next day, cells were stimulated with 50 ng/ml of TNFα for the given timepoints and washed once with PBS. Nuclear (N) and cytoplasmic (C) fractions were separated using the Pierce NE-PER Kit and assayed via immunoblotting with the indicated antibodies. Quantification (Quant) of nuclear pS276 p65 was calculated by dividing the densitometric value of each lane by the corresponding nuclear HDAC1 value. Results are representative of three experiments.

Article Snippet: Anti-p65 pS276 (3027) and anti-pIKKα/pIKKβ (2681) antibodies were from Cell Signaling (Danvers, MA).

Techniques: Immunofluorescence, Staining, Western Blot

Journal: Oncogene

Article Title: The Prolyl Isomerase Pin1 Regulates the NF-κB Signaling Pathway and Interleukin-8 Expression in Glioblastoma

doi: 10.1038/onc.2009.232

Figure Lengend Snippet: (A) U251-TR.sh-p65 cells were grown in the absence or presence of Tet for 48 h, serum-starved, and then stimulated with TNFα (10 ng/ml) for 4 h. Total RNA was isolated, first-strand cDNA synthesis was performed, and RT-PCR was performed on the samples using primers to specifically detect p65, IL-8 and GAPDH. (B) U251-TR.sh-p65 cells were grown in the absence or presence of Tet for 48 h, serum-starved, and the stimulated with TNFα (10 ng/ml) for 24 h. Supernatant was collected and analyzed by ELISA for IL-8 protein expression (*p<0.05). (C) U251-TR.sh-p65 cells were grown in the absence or presence of Tet for 48 h, serum-starved, and then stimulated with TNFα (10 ng/ml) for 15 min. The samples were then subjected to ChIP assay (see Materials and Methods). Immunoprecipitations were performed with anti-p65 or an IgG control. All results are representative of three experiments.

Article Snippet: Anti-p65 pS276 (3027) and anti-pIKKα/pIKKβ (2681) antibodies were from Cell Signaling (Danvers, MA).

Techniques: Isolation, cDNA Synthesis, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Expressing, Control

Journal: Journal of Cancer

Article Title: Properties of Lewis Lung Carcinoma Cells Surviving Curcumin Toxicity

doi: 10.7150/jca.3659

Figure Lengend Snippet: Evaluation of cancer stem cell markers by ELISA

Article Snippet: The plates were then washed with PBS twice and incubated with 100 μl complete DMEM containing rabbit polyclonal antibodies (anti-p65, Santa Cruz Biotechnology, SC-109, 200 µg/ml and anti-ALDH1A1, Proteintech Group, 15910-1-AP, 133 µg/ml) at 37°C for two hours.

Techniques: