NF-κB inhibitor effect on lipopolysaccharide-induced monocyte chemoattractant protein-1 and osteopontin expression in NZB/W mesangial cells

<p><b>Copyright information:</b></p><p>Taken from "Mesangial cells of lupus-prone mice are sensitive to chemokine production"</p><p>http://arthritis-research.com/content/9/4/R67</p><p>Arthritis Research & Therapy 2007;9(4):R67-R67.</p><p>Published online 7 Jul 2007</p><p>PMCID:PMC2206365.</p><p></p> Growth-arrested (under 2% fetal bovine serum (FBS)) mesangial cells were preincubated for 2 hours with -tosyl-1-phenylalanine chloromethyl ketone (TPCK) or dexamethasone (Dex), and then 10 μg/ml lipopolysaccharide (LPS) was added for 12 hours: monocyte chemoattractant protein-1 (MCP-1) mRNA levels and osteopontin (OPN) mRNA levels were measured by real-time PCR analysis. Growth-arrested (under 2% FBS) mesangial cells were preincubated for 2 hours with TPCK or Dex, then 10 μg/ml LPS was added for 24 hours: MCP-1 protein levels in the supernatant were measured by ELISA, and OPN protein levels in the cell lysate were measured by western blot analysis; semiquantitative data are shown. The MCP-1 protein expression levels at various time points were normalized to total protein (pg/mg). The experiment was performed in triplicate, and results are expressed as the mean ± standard error. **< 0.01, ***< 0.005, = 6. White bar, absence of LPS; black bar, LPS alone; hatched bar, TPCK and LPS or Dex and LPS

Monocyte chemoattractant protein-1 and osteopontin expression in NZB/W mesangial cells with lipopolysaccharide treatment

<p><b>Copyright information:</b></p><p>Taken from "Mesangial cells of lupus-prone mice are sensitive to chemokine production"</p><p>http://arthritis-research.com/content/9/4/R67</p><p>Arthritis Research & Therapy 2007;9(4):R67-R67.</p><p>Published online 7 Jul 2007</p><p>PMCID:PMC2206365.</p><p></p> Growth-arrested (under 2% fetal bovine serum (FBS)) mesangial cells were incubated with 10 μg/ml lipopolysaccharide (LPS) for different periods of time, and then levels of monocyte chemoattractant protein-1 (MCP-1) mRNA or osteopontin (OPN) mRNA were examined by real-time PCR. MCP-1 protein levels in the culture supernatant were measured by ELISA. OPN protein levels in the cell lysate were measured by western blot analysis; semiquantitative data are shown. The MCP-1 protein expression levels at various time points were normalized to total protein (pg/mg). The experiment was performed in triplicate, and results are expressed as the mean ± standard error, = 6. The last time point without LPS stimulation (unstimulated) was presented as the negative control. *< 0.05, **< 0.01, ***< 0.005

Toll-like receptor 4 and myeloid differentiation factor 88 mRNA in NZB/W mesangial cells (lipopolysaccharide treatment)

<p><b>Copyright information:</b></p><p>Taken from "Mesangial cells of lupus-prone mice are sensitive to chemokine production"</p><p>http://arthritis-research.com/content/9/4/R67</p><p>Arthritis Research & Therapy 2007;9(4):R67-R67.</p><p>Published online 7 Jul 2007</p><p>PMCID:PMC2206365.</p><p></p> Growth-arrested (under 2% fetal bovine serum (FBS)) mesangial cells were incubated with 10 μg/ml lipopolysaccharide (LPS) for different periods of time, and then Toll-like receptor 4 (TLR-4) and myeloid differentiation factor 88 (MyD88) mRNA levels were measured by real-time PCR analysis. The experiment was performed in triplicate, and results are expressed as the mean ± standard error, = 6. The last time point without LPS stimulation (unstimulated) was presented as the negative control. *< 0.05, **< 0.01

NF-κB p65 activation in NZB/W mesangial cells with lipopolysaccharide treatment

<p><b>Copyright information:</b></p><p>Taken from "Mesangial cells of lupus-prone mice are sensitive to chemokine production"</p><p>http://arthritis-research.com/content/9/4/R67</p><p>Arthritis Research & Therapy 2007;9(4):R67-R67.</p><p>Published online 7 Jul 2007</p><p>PMCID:PMC2206365.</p><p></p> Growth-arrested (under 2% fetal bovine serum (FBS)) mesangial cells (MCs) were incubated with 10 μg/ml lipopolysaccharide (LPS) for different periods of time, and then the distribution of NF-κB p65 was examined. Immunofluorescence: images a–d, NZB/W MCs; images e–h, DBA/W MCs (nonimmune strain, served as control) incubated for 0–12 hours with LPS; and image i, semiquantitative data. Electrophoresis mobility-shift assay performed using a DIG-labeled synthetic oligonucleotide and nuclear extract from MCs. The competition assay used the same unlabeled oligonucleotide at a 10-fold higher concentration. Arrow, NF-κB p65 binding bands. Comp., the abbreviation of competition. ELISA performed using the TransAM NF-κB p65 kit. The NF-κB p65 expression levels at various time points were normalized to nuclear protein (ng/mg). The experiment was performed in triplicate, and results are expressed as the mean ± standard error, = 6. The last time point without LPS stimulation (unstimulated) was presented as the negative control. ***< 0.005 versus DBA/W MC controls

Modified Salicylanilide and 3‑Phenyl‑2<i>H</i>‑benzo[<i>e</i>][1,3]oxazine-2,4(3<i>H</i>)‑dione Derivatives as Novel Inhibitors of Osteoclast Differentiation and Bone Resorption

Inhibition of osteoclast formation is a potential strategy to prevent inflammatory bone resorption and to treat bone diseases. In the present work, the purpose was to discover modified salicylanilides and 3-phenyl-2<i>H</i>-benzo­[<i>e</i>]­[1,3]­oxazine-2,4­(3<i>H</i>)-dione derivatives as potential antiosteoclastogenic agents. Their inhibitory effects on RANKL-induced osteoclastogenesis from RAW264.7 cells were evaluated by TRAP stain assay. The most potent compounds, <b>1d</b> and <b>5d,</b> suppressed RANKL-induced osteoclast formation and TRAP activity dose-dependently. The cytotoxicity assay on RAW264.7 cells suggested that the inhibition of osteoclastic bone resorption by these compounds did not result from their cytotoxicity. Moreover, both compounds downregulated RANKL-induced NF-κB and NFATc1 in the nucleus, suppressed the expression of osteoclastogenesis-related marker genes during osteoclastogenesis, and prevented osteoclastic bone resorption but did not impair osteoblast differentiation in MC3T3-E1. Therefore, these modified salicylanilides and 3-phenyl-2<i>H</i>-benzo­[<i>e</i>]­[1,3]­oxazine-2,4­(3<i>H</i>)-diones could be potential lead compounds for the development of a new class of antiresorptive agents

SNPs of the <i>IL10</i> gene cluster associated with SLE in European Americans.

<p>(A) Association of 19 genotyped SNPs with SLE in EA (red), AA (yellow), AS (blue) and HS (green). Allelic <i>P</i> value (−log₁₀<i>P</i>) of each SNP was plotted against its genomic position. (B) Association of 19 genotyped and an additional 109 imputed SNPs with SLE in EA. Genotyped and imputed SNPs were indicated as circles and triangles, respectively. Based on its pairwised LD strength with rs3024505, each SNP was highlighted as red (r²>0.9) or grey (r²<0.9). (C) Genomic structure of the <i>IL10</i> gene cluster and the location of each SNP. (D) Haplotypic analysis in EA. Haplotypes were constructed using four SLE-associated SNPs shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003870#pgen-1003870-g001" target="_blank">Figure 1B</a> (rs3024505, rs3024495, rs3024493 and rs3122605), three previously reported SLE-associated SNPs (rs1800872, rs1800871 and rs1800896 in the promoter of <i>IL10</i>) and rs1518111 (the T allele associated with Bechet's disease). Risk alleles of four SLE-associated SNPs shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003870#pgen-1003870-g001" target="_blank">Figure 1B</a> were bolded and italicized.</p

Dose-dependent association of rs3122605 risk G-allele with elevated levels of <i>IL10</i> mRNA and protein.

<p><i>IL10</i> mRNA (A) and protein levels (B) were measured in PBMCs and plasma from EA SLE patients and healthy controls, respectively. Each symbol represents an individual and horizontal lines indicate mean ± SEM values.</p