Journal: Mediators of Inflammation

Article Title: MCL Plays an Anti-Inflammatory Role in Mycobacterium tuberculosis -Induced Immune Response by Inhibiting NF- κ B and NLRP3 Inflammasome Activation

doi: 10.1155/2017/2432904

Figure Lengend Snippet: MCL inhibits Mtb-triggered NF- κ B activation. Raw264.7 cells (1 × 10 6 /well) were seeded in 6-well plates and infected with Mtb for 4 hrs. Cells were washed by PBS and treated with MCL (10 μ M) for indicated time courses. Phosphorylation of p65 and β -actin was detected by Western blot (a). The lower bar graph showed the statistical results for phosphorylation levels of NF- κ B p65 (a). Data are presented as mean ± SD in at least three independent experiments, ∗∗ p < 0.01. Raw264.7 cells (3 × 10 5 /well) were cotransfected with NF- κ B luciferase reporter plasmid and pRL-TKR Renilla luciferase plasmid as previous study . After 30 hr, cells were infected with H37Ra at MOI of 10 and treated with MCL (10 μ M) for 12 hr, and the luciferase activities were measured (b). Data are shown with the means ± SD ( n ≧3). ∗ p < 0.05. Raw264.7cells (3 × 10 5 /well) were plated in climbing slice and followed by appropriate treatment. Immunofluorescence assay was conducted with rabbit anti-pp65 antibody (green) and DAPI (blue) (c). The results represented three independent experiments.

Article Snippet: Rabbit anti-ASC (10500-1-AP, proteintech), anti-LC3 (sc-16755, Santa Cruz Biotechnology), anti-caspase-1 (sc-514, Santa Cruz Biotechnology), and anti-pp65 (cst-3033, cell signaling technology) antibodies were used for immunoblot.

Techniques: Activation Assay, Infection, Western Blot, Luciferase, Plasmid Preparation, Immunofluorescence

Journal: International journal of obesity (2005)

Article Title: Hypoxia reduces the response of human adipocytes towards TNFα resulting in reduced NF-κB signaling and MCP-1 secretion.

doi: 10.1038/ijo.2011.200

Figure Lengend Snippet: Figure 4. Expression of NF-kB signaling proteins under hypoxia and phosphorylation kinetics of NF-kB and IkBa during TNFa stimulation. Differentiated human primary adipocytes were incubated for 8 h under normoxia or 1% hypoxia. Afterwards, cells were treated with 50 ng TNFa and directly lysed at the indicated time points. Total lysates (10 mg) were analyzed by western blotting as described in Figure 1 (a) Expression level of NF-kB, IkBa, IKKa and IKKb after 8 h of normoxia or hypoxia. (b, c) Kinetic of NF-kB and IkBa phosphorylation, shown as the rise according to basal normoxic or hypoxic phosphorylation. (d) Phosphorylation of NF-kB after 10 min of stimulation with different concentrations of TNFa. Representative blots are shown. Data sets are mean values±s.e.m. of 4--10 independent experiments normalized to actin. *Po0.05 compared with the normoxic value.

Article Snippet: Human specific anti-phospho-NF-kB (pp65) (Ser536), anti-NF-kB (p65), anti-IkBa and anti-phospho-IkBa (Ser32) antibodies were supplied by Cell Signaling Technology (Frankfurt, Germany).

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

Journal: bioRxiv

Article Title: Tumor immune microenvironment permissive to metastatic progression of ING4-deficient breast cancer

doi: 10.1101/2024.05.09.593418

Figure Lengend Snippet: (A) Immunohistochemical staining images of tumors positive for: a) ING4, b) pp65/RelA(Ser276), c) CD68, d) CD4, e) CD8, f) PD-1; black boxes in the top panel are presented in higher magnification images in the bottom panel; black scale bars denote 100 μm in length. (B) Percentage of tumors positive for each marker (solid dark bar): ING4 (154/246, 63%), pp65 (84/245, 34%), CD68 (171/193, 89%), CD4 (50/193, 26%), CD8 (75/189, 40%), and PD-1 (24/132, 18%). (C) Venn diagram and table showing the number of tumors positive for CD68, CD8, CD4, and/or PD-1. (D) No association between immune markers with ING4-low vs ING4-high (left graph) or NF-κB-low vs NF-κB-high (right graph) tumors. (E) Increased CD8+ tumors in ING4-low/pp65-high tumors (solid dark bar, 59% vs 32-38%); p values were determined by Fisher’s Exact test; ns , not significant. (F) High lymph node positivity in ING4-low/pp65-high tumors (LN+, solid dark bar): All, all tumors; ER-, estrogen receptor-negative.

Article Snippet: Antibodies used in Western blots were against: ING4 (BTIM-4 hybridoma supernatant, 1:4 [ ]), pp65/RelA (Ser536) (93H1, 1:1,000; CST), histone H3 (1:5000, CST), and α-tubulin (DM1A, 1:5000, Sigma-Aldrich).

Techniques: Immunohistochemical staining, Staining, Marker

Journal: bioRxiv

Article Title: Tumor immune microenvironment permissive to metastatic progression of ING4-deficient breast cancer

doi: 10.1101/2024.05.09.593418

Figure Lengend Snippet: (A) Schematic diagram of the Ing4 genome with exons (open boxes); CRISPR/CAS9 gRNAs R1 and M1 mapped to exon 1 and exon 3 (red line). (B) Nuclear accumulation of pp65/RelA in Ing4 -deleted p53MT cells treated with cytokines: v2, the vector control cells; v2R1, R1 gRNA construct cells; v2M1, M1 gRNA construct cells; cells were treated with PBS (-), 10ng/ml TNFα (T), or 10ng/ml IL-1β (I), for 1 hour in serum-free media prior to cell fractionation; nuc, nuclear fraction; cyto, cytoplasmic fraction; Western blot for ING4 and phospho-p65/RelA (ser536); Histone H3 and α-tubulin antibodies were used as the loading controls. (C) Relative fold expression of the mouse Il6 gene in p53MT cells treated with (+) or without (-) TNFα (left panel) or IL-1β (right panel) for 4 hours in serum-free media: open bar, v2; closed bar, v2R1; serrated bar, v2M1; RT-qPCR to quantify Il6 expression using Gapdh as the reference; p values were determined by two-tailed student t-test; ns , not significant. (D) Cytokine-induced migration of Ing4 -deleted p53MT cells: PBS (-),TNFα (T), or IL-1β (I); v2, v2R1, or v2M1 cells migrated through 8μm pores in the transwell inserts were stained with DAPI and visualized under a fluorescent microscope. Cell numbers were determined by averaging a minimum of 4 images per experiment from at least 3 independent experiments; p values were determined by two-tailed student t-test; *, p < 0.001; **, p < 0.05.

Article Snippet: Antibodies used in Western blots were against: ING4 (BTIM-4 hybridoma supernatant, 1:4 [ ]), pp65/RelA (Ser536) (93H1, 1:1,000; CST), histone H3 (1:5000, CST), and α-tubulin (DM1A, 1:5000, Sigma-Aldrich).

Techniques: CRISPR, Plasmid Preparation, Control, Construct, Cell Fractionation, Western Blot, Expressing, Quantitative RT-PCR, Two Tailed Test, Migration, Staining, Microscopy

Journal: Journal of Translational Medicine

Article Title: Transplanted allogeneic cardiac progenitor cells secrete GDF-15 and stimulate an active immune remodeling process in the ischemic myocardium

doi: 10.1186/s12967-022-03534-0

Figure Lengend Snippet: Antibody for flow cytometery

Article Snippet: pP65 , 4886 , Cellsignalling , FITC.

Techniques: Blocking Assay

Journal: Journal of Translational Medicine

Article Title: Transplanted allogeneic cardiac progenitor cells secrete GDF-15 and stimulate an active immune remodeling process in the ischemic myocardium

doi: 10.1186/s12967-022-03534-0

Figure Lengend Snippet: Role of GDF15 in cardiac recovery. Basal protein expression of GDF15 in rat CPCs and secretome ( A ), GDF15 protein expression in rCPCs GDF15KD and rCPCs scramble ( B ). Cardiac functional parameters were measured after intramyocardial injection of one million rCPCs, rCPCs GDF15KD and placebo separately in the BN rat MI model. C LV ejection fraction, D LV fractional shortening. Representative picture ( E ) and quantification ( F ) of Masson trichrome staining in rat hearts. Serum ELISAs for the cytokines IL-10, IL-2, and TNF- α are shown in G , Single-cell suspensions of total heart lysates were used for flow cytometric analysis 5 days after intramyocardial injection of one million rCPCs, rCPCS GDF15KD , or placebo in BN rats. Flow images ( H ) and quantitative flow results for Tregs ( I ). Flow images ( J ) and quantitative flow results ( K ) for M2 cells. Phosphorylated NF-κB p65 flow images ( L ) and quantification ( M ). Whole hearts were also obtained on day 5 for immunohistochemistry studies. Images of phosphorylated NF-κB, FoxP3 + , and DAPI staining ( N ) and quantification ( O ). Immunohistochemistry of phosphorylated NF-κB and sarcomeric actin with DAPI ( P ). Numerical data are summarized as box and whisker plots with a median value (black bar inside box), 25th and 75th percentiles (bottom and top of box, respectively), and minimum and maximum values (bottom and top whisker, respectively). The number (n) of rats in each group indicated near the (up/below/on) each respective box and whisker plot. Q Invitro co-culture assay with rCPCs/ rCPCS GDF15KD with BN rat spleenocytes.for 5 day and CD48 measured by FACS

Article Snippet: pP65 , 4886 , Cellsignalling , FITC.

Techniques: Expressing, Functional Assay, Injection, Staining, Immunohistochemistry, Whisker Assay, Co-culture Assay