Journal: Biochemical and biophysical research communications

Article Title: Platelet-derived high-mobility group box 1 promotes recruitment and suppresses apoptosis of monocytes

doi: 10.1016/j.bbrc.2016.07.078

Figure Lengend Snippet: HMGB1 inhibits monocyte apoptosis induced by ABT-737 (A,C) or staurosporine (B,D), which is reversed when monocytes are pretreated with a blocking TLR4 antibody, as evaluated by Annexin V (A,B) and TMRE (C,D) stainings. (E) U0126, a specific MEK/ERK inhibitor, inhibits the effect of rHMGB1 on TMRE fluorescence in monocytes. (F) rHMGB1 (100 ng/ml) induces phosphorylation of ERK in monocytes, which does not occur when monocytes are pretreated with a blocking TLR4 antibody. Data are presented as mean ± SD for N≥4 and at least three separate experiments in all studies. * p<0.05, # p<0.05, ** p<0.01 (Student’s t test).

Article Snippet: When indicated, HMGB1 receptors were blocked on monocytes with anti-human RAGE polyclonal antibody (20 μg/ml, goat IgG), anti-human TLR2 monoclonal antibody (2 μg/ml, mouse IgG2b) or anti-human TLR4 polyclonal antibody (10 μg/ml, goat IgG) (R&D Systems, Wiesbaden, Germany).

Techniques: Blocking Assay, Fluorescence, Phospho-proteomics

Journal: Journal of Leukocyte Biology

Article Title: CD68 on rat macrophages binds tightly to S100A8 and S100A9 and helps to regulate the cells’ immune functions

doi: 10.1189/jlb.2a0415-170rrr

Figure Lengend Snippet: Figure 8. Inhibitory effects of specific antibodies for CD68, TLR4, and RAGE on the expression of mRNAs for S100A8 and S100A9 in macrophages. We measured the mRNA expression levels of S100A8 and S100A9 to investigate the effects of specific antibodies for CD68, TLR4, and RAGE on the immune functions of macrophages that had been activated with r-S100A8 (10 mg/ml) or r-S100A9 (10 mg/ml) using a StepOnePlus Real-Time PCR System. (A) RT-PCR was carried out to detect S100A8 mRNA in macrophages that had been activated with r-S100A8 (10 mg/ml) or r-S100A9 (10 mg/ml) for 0.5 h in 5% CO2 at 37°C in the presence and absence of each antibody. (B) RT-PCR was also carried out to detect S100A9 mRNA in macrophages that had been activated with r-S100A8 (10 mg/ml) or r-S100A9 (10 mg/ml) alone for 0.5 h in 5% CO2 at 37°C in the presence and absence of each antibody. Error bars represent the SD (n = 3) of independent experiments. In this study, the experimental procedures have been repeated 3 times. Statistical analysis was performed using Dunnett’s test; *P , 0.05.

Article Snippet: Anti-TLR4 mAb was obtained from Novus Biologicals (Littleton, CO, USA; NB100-56566).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Leukocyte Biology

Article Title: CD68 on rat macrophages binds tightly to S100A8 and S100A9 and helps to regulate the cells’ immune functions

doi: 10.1189/jlb.2a0415-170rrr

Figure Lengend Snippet: Figure 9. Inhibitory effects of specific antibodies for CD68, TLR4, and RAGE on the expression of mRNAs for inflammatory and anti-inflammatory cytokines in macrophages. We measured the mRNA expression levels of IL-1b, TNF-a, IL-6, IL- 10, and TGF-b to investigate the effects of specific antibodies for CD68, TLR4, and RAGE on the immune functions of macrophages that had been activated with r-S100A8 (10 mg/ml) or r-S100A9 (10 mg/ml) alone using a StepOnePlus Real-Time PCR System. (A–E) RT-PCR was carried out to detect these cytokine mRNAs in macrophages. Changes in the mRNA expression levels of IL-1b (Α), TNF-a (Β), IL-6 (C), IL-10 (D), and TGF-b (E), respectively, in macrophages that had been activated with r-S100A8 (10 mg/ml) or r-S100A9 (10 mg/ml) alone for 0.5 h in 5% CO2 at 37°C in the presence and absence of each antibody. Error bars represent the SD (n = 3) of independent experiments. In this study, the experimental procedures have been repeated 3 times. Statisti- cal analysis was performed using Dunnett’s test; *P , 0.05.

Article Snippet: Anti-TLR4 mAb was obtained from Novus Biologicals (Littleton, CO, USA; NB100-56566).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

Journal: The American Journal of Pathology

Article Title: Enhanced Susceptibility to Endotoxic Shock and Impaired STAT3 Signaling in CD31-Deficient Mice

doi: 10.1016/s0002-9440(10)62243-2

Figure Lengend Snippet: Figure 11. TLR4 expression of WT and CD31-deficient splenocytes and endothelial cells remains constant and does not change following LPS treat- ment. Representative FACS analyses of WT (A) and KO (B) splenocytes in the absence and presence of LPS; KO (C) and PECAM-1 reconstituted (D) lung endothelial cells and KO (E) and WT (F) brain endothelial cells illus- trating essentially no differences in TLR4 expression. This was confirmed by Western blot analyses (data not shown).

Article Snippet: Splenocytes were prepared as described, fixed in 2% PFA, then stained with 0.5 g of polyclonal anti-mouse TLR4 (Imgenex) in Hanks’ buffered salt solution plus 1% BSA for 1 hour on ice, then, following two washes, in donkey anti-rabbit IgG F(ab )2 PE conjugate (Jackson Immunoresearch, West Grove, PA).

Techniques: Expressing, Western Blot

Journal: Infection and Immunity

Article Title: Cell Activation by Ligands of the Toll-Like Receptor and Interleukin-1 Receptor Family Depends on the Function of the Large-Conductance Potassium Channel MaxiK in Human Macrophages

doi: 10.1128/iai.01783-05

Figure Lengend Snippet: FIG. 3. Colocalization of MaxiK and TLR4 determined by confocal microscopy. HEK293-TLR4/MD-2-MaxiKGFP cells were subjected to time lapse experiments before (left) and 3 min after (right) stimulation with 10 ng/ml LPS. Cells were placed on a temperature-adjustable microscope stage and equilibrated to 37°C. In unstimulated cells, TLR4 (yellow) and MaxiK (blue) localize to different compartments. Upon stimulation with LPS, distinct areas where TLR4 and MaxiK colocalize can be observed (white pixels in the image). Lower panels are enlargements of boxed areas. Bars, 10 M. Results shown are from one experiment representative of three independent experiments.

Article Snippet: Briefly, HEK293-TLR4/MD2-MaxiKGFP cells were cultured on microchamber culture slides and stained with an anti-TLR4 antibody (HTA125; Imgenex) and an Alexa 647- conjugated goat anti-mouse secondary antibody (Molecular Probes, Oregon).

Techniques: Confocal Microscopy, Microscopy

Journal: Kidney international

Article Title: Poly(ADP-ribose) polymerase 1 activation is required for cisplatin nephrotoxicity.

doi: 10.1038/ki.2012.64

Figure Lengend Snippet: Figure 5 | Poly(ADP-ribose) polymerase 1 (Parp1) deficiency inhibits inflammation induced by cisplatin injection in kidneys. The kidneys in Parp1-knockout (KO) and -wild-type (WT) male mice were harvested 0, 1, 2, 3, or 5 days after cisplatin (20 mg/kg body weight) or saline (control) injection. (a) Polymorphonuclear neutrophil (PMN)–positive cells were counted in 10 fields ( 200 magnification) per kidney (n ¼ 4 in each group). (b) The expression of intercellular adhesion molecule-1 (ICAM-1), tumor necrosis factor-a (TNF-a), and Toll-like receptor 4 (TLR4) in kidneys was examined by western blot analysis. Anti-b-actin antibody was used as a loading control. (c–e) The intensities of protein bands (ICAM-1, 110 kDa; TNF-a, 26 kDa; TLR4, 90 kDa; all approximate) were quantified using the Lab Works analysis software (n ¼ 6 in each group). *Po0.05 vs. 0 days, #Po0.05 vs. WT.

Article Snippet: Western blot analysis was confirmed, as described previously,62–64 using antibodies against PARP1, cleaved caspase-3, phospho-RelA, phospho-p38, phospho-38, phospho-JNK, JNK, phospho-MKK3/6, phospho-MKK4 (Cell Signaling, Beverly, MA), ICAM-1, RelA, IkBa (Santa Cruz, Santa Cruz, CA), PAR (BD Pharmingen, San Jose, CA), b-actin (Sigma, St. Louis, MO), TNF-a (Abcam, Cambridge, MA), or TLR4 (Imgenex, San Diego, CA).

Techniques: Injection, Knock-Out, Saline, Control, Expressing, Western Blot, Software

Journal: Kidney international

Article Title: Poly(ADP-ribose) polymerase 1 activation is required for cisplatin nephrotoxicity.

doi: 10.1038/ki.2012.64

Figure Lengend Snippet: Figure 8 | Poly(ADP-ribose) polymerase 1 (Parp1) deficiency blocks TLR4/MAPK/NF-jB/TNF-a signaling pathway during necrotic cell death induced by cisplatin treatment in primary culture of proximal tubule epithelial cells. The proximal tubule epithelial cells were isolated from Parp1-knockout (KO) and -wild-type (WT) mouse kidneys. After 18 h of starvation, the cells were treated with 400 mmol/l cisplatin for 4 h. Dimethyl sulfoxide (DMSO) was used as vehicle. To downregulate Toll-like receptor 4 (TLR4) expression, 500 pmol of small interference RNA (siRNA) was transfected on a 100-mm culture dish for 24 h before cisplatin or vehicle treatment. The same concentration of scramble siRNA was used as control. Pharmacological inhibitors (100 mmol/l of SB203580 against p38 activation, 10 mmol/l of SP600125 against c-Jun N-terminal kinase (JNK) activation, 100 mmol/l of ammonium pyrrolidinedithiocarbamate against nuclear factor-kB (NF-kB) activation, or 100 mmol/l of thalidomide against tumor necrosis factor-a (TNF-a) synthesis) or the same volume of DMSO (control) were added 2 h before cisplatin or vehicle treatment. Results are representative of experiments repeated three times.

Article Snippet: Western blot analysis was confirmed, as described previously,62–64 using antibodies against PARP1, cleaved caspase-3, phospho-RelA, phospho-p38, phospho-38, phospho-JNK, JNK, phospho-MKK3/6, phospho-MKK4 (Cell Signaling, Beverly, MA), ICAM-1, RelA, IkBa (Santa Cruz, Santa Cruz, CA), PAR (BD Pharmingen, San Jose, CA), b-actin (Sigma, St. Louis, MO), TNF-a (Abcam, Cambridge, MA), or TLR4 (Imgenex, San Diego, CA).

Techniques: Isolation, Knock-Out, Expressing, Transfection, Concentration Assay, Control, Activation Assay

Journal: Kidney international

Article Title: Poly(ADP-ribose) polymerase 1 activation is required for cisplatin nephrotoxicity.

doi: 10.1038/ki.2012.64

Figure Lengend Snippet: Figure 11 | Poly(ADP-ribose) polymerase 1 (PARP1) inhibitor reduces inflammation induced by cisplatin injection in kidneys. The kidneys in 129S1/SvImJ male mice were harvested 0, 3, or 5 days after cisplatin injection. The mice were administered either PJ34 (10 mg/kg body weight) or saline (control, Con) twice daily intraperitoneally from 24 h before cisplatin injection up to the time that they were killed. (a) Polymorphonuclear neutrophil (PMN)-positive cells were counted in 10 fields ( 200 magnification) per kidney (n ¼ 4 in each group). (b) The expression of Poly(ADP-ribose) (PAR), intercellular adhesion molecule-1 (ICAM-1), tumor necrosis factor-a (TNF-a), and Toll-like receptor 4 (TLR4) in kidneys was examined by western blot analysis. Anti-b-actin antibody was used as a loading control. (c–f) The intensities of protein bands (PAR, 116 kDa; ICAM-1, 110 kDa; TNF-a, 26 kDa; TLR4, 90 kDa; all approximate) were quantified using the Lab Works analysis software (n ¼ 4 in each group). *Po0.05 vs. 0 days; #Po0.05 vs. control.

Article Snippet: Western blot analysis was confirmed, as described previously,62–64 using antibodies against PARP1, cleaved caspase-3, phospho-RelA, phospho-p38, phospho-38, phospho-JNK, JNK, phospho-MKK3/6, phospho-MKK4 (Cell Signaling, Beverly, MA), ICAM-1, RelA, IkBa (Santa Cruz, Santa Cruz, CA), PAR (BD Pharmingen, San Jose, CA), b-actin (Sigma, St. Louis, MO), TNF-a (Abcam, Cambridge, MA), or TLR4 (Imgenex, San Diego, CA).

Techniques: Injection, Saline, Control, Expressing, Western Blot, Software