Effect of Ras/Raf-1 modulation on transforming growth factor-beta-1 (TGF-β1)-induced expression of the gene

<p><b>Copyright information:</b></p><p>Taken from "Inorganic pyrophosphate generation by transforming growth factor-beta-1 is mainly dependent on ANK induction by Ras/Raf-1/extracellular signal-regulated kinase pathways in chondrocytes"</p><p>http://arthritis-research.com/content/9/6/R122</p><p>Arthritis Research & Therapy 2007;9(6):R122-R122.</p><p>Published online 22 Nov 2007</p><p>PMCID:PMC2246241.</p><p></p> Rat chondrocytes were electroporated with empty vector, wild-type, constitutively active, or dominant-negative plasmids for Ras or Raf-1 (2 μg/well of six-well plate) before stimulation with 10 ng/mL of TGF-β1 for 12 hours. Total RNA was extracted and subjected to real-time polymerase chain reaction analysis. The mRNA level of Ank was normalized to that of S29 mRNA and is expressed as mean percentages (± standard deviation) over control values from three independent experiments. Statistically significant differences from the control are indicated as *< 0.05 and from TGF-β1-treated cells as #< 0.05. Effect of TGF-β1 on extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in electroporated cells with wild-type, constitutively active, or dominant-negative plasmids for Ras (2 μg/well of six-well plate). Total proteins were extracted from rat chondrocytes exposed to 10 ng/mL of TGF-β1 for 15 minutes and subjected to Western blotting using anti-phospho- and anti-total-ERK 1/2 antibodies. The relative abundance of these proteins was normalized to that of β-actin protein

Effect of transforming growth factor-beta-1 (TGF-β1) on proteins regulating inorganic pyrophosphate metabolism

<p><b>Copyright information:</b></p><p>Taken from "Inorganic pyrophosphate generation by transforming growth factor-beta-1 is mainly dependent on ANK induction by Ras/Raf-1/extracellular signal-regulated kinase pathways in chondrocytes"</p><p>http://arthritis-research.com/content/9/6/R122</p><p>Arthritis Research & Therapy 2007;9(6):R122-R122.</p><p>Published online 22 Nov 2007</p><p>PMCID:PMC2246241.</p><p></p> Phenotypic characterization of chondrocytes. Total RNA was extracted from untreated rat chondrocytes and subjected to real-time polymerase chain reaction (PCR) analysis. The relative abundance of gene mRNAs was normalized to that of S29 mRNA. Results are presented in histograms as mean percentages (± standard deviation [SD]) over S29 value. Effect of TGF-β1 on Ank, PC-1, and TNAP mRNA levels. Total RNA was extracted from rat chondrocytes exposed to 10 ng/mL of TGF-β1 from 1 to 48 hours and subjected to real-time PCR analysis. The relative abundance of gene mRNAs was normalized to that of S29 mRNA. Results are expressed as mean percentages (± SD) over control values. Statistically significant differences from the control are indicated as *< 0.05. Effect of TGF-β1 on ANK or PC-1 protein levels. Total proteins were extracted from rat chondrocytes exposed to 10 ng/mL of TGF-β1 from 6 to 48 hours and subjected to Western blotting using polyclonal anti-ANK and anti-PC-1 antibody. The protein band intensities were quantified by densitometry from enhanced chemiluminescence immunoblots. The relative abundance of these proteins was normalized to that of β-actin protein and expressed as induction folds over control value. N.D., not detected; TNAP, tissue-nonspecific alkaline phosphatase

Effect of Smad 7 overexpression on transforming growth factor-beta-1 (TGF-β1)-induced responses in rat chondrocytes

<p><b>Copyright information:</b></p><p>Taken from "Inorganic pyrophosphate generation by transforming growth factor-beta-1 is mainly dependent on ANK induction by Ras/Raf-1/extracellular signal-regulated kinase pathways in chondrocytes"</p><p>http://arthritis-research.com/content/9/6/R122</p><p>Arthritis Research & Therapy 2007;9(6):R122-R122.</p><p>Published online 22 Nov 2007</p><p>PMCID:PMC2246241.</p><p></p> Rat chondrocytes were electroporated with either empty vector or wild-type Smad 7 overexpressing plasmid (2 μg/well of six-well plate) and then treated for 12 hours with 10 ng/mL of TGF-β1. Total RNA was extracted and subjected to real-time polymerase chain reaction analysis. The mRNA level of aggrecan and Ank was normalized to that of S29 mRNA and is expressed as mean percentages (± standard deviation) over control values from three independent experiments. Statistically significant differences from the control are indicated as *< 0.05 and from TGF-β1-treated cells as #< 0.05

Respective contributions of and to transforming growth factor-beta-1 (TGF-β1)-induced increase in extracellular inorganic pyrophosphate (ePPi) production

<p><b>Copyright information:</b></p><p>Taken from "Inorganic pyrophosphate generation by transforming growth factor-beta-1 is mainly dependent on ANK induction by Ras/Raf-1/extracellular signal-regulated kinase pathways in chondrocytes"</p><p>http://arthritis-research.com/content/9/6/R122</p><p>Arthritis Research & Therapy 2007;9(6):R122-R122.</p><p>Published online 22 Nov 2007</p><p>PMCID:PMC2246241.</p><p></p> Effect of small interfering RNA (siRNA) on Ank and PC-1 mRNA levels. Rat chondrocytes were transfected with siRNA 24 hours before TGF-β1 stimulation. Total RNA was extracted from rat chondrocytes exposed to 10 ng/mL of TGF-β1 for 12 hours (Ank) or 24 hours (PC-1) and then subjected to real-time polymerase chain reaction analysis. The level of Ank, PC-1, and L27 mRNAs was normalized to that of S29 mRNA and expressed as mean percentages (± SD) over control values. Effect of Ank or PC-1 siRNA on ePPi levels. Shown are levels of ePPi in culture supernatant of rat chondrocytes transfected with siRNA and then stimulated for 12 or 24 hours with 10 ng/mL of TGF-β1. ePPi levels were normalized to the amount of total cell proteins (= 6) and are expressed as mean (± SD) in picomoles per microgram of protein. Statistically significant differences from the control are indicated as *< 0.05 and from TGF-β1-treated cells as #< 0.05

Effect of rosiglitazone and pioglitazone treatment on histological grading of ankle lesions during adjuvant-induced arthritis

Animals were treated daily for 21 days with rosiglitazone (ROSI) 10 mg/kg (= 8) or pioglitazone (PIO) 30 mg/kg (= 8) by oral administration. Arthritic (adjuvant-induced arthritis (AIA)) (= 7) and normal controls (= 7) were given 0.5% carboxymethylcellulose alone. Cartilage degradation and bone erosion were graded as indicated in the Materials and methods section. Data are expressed as means ± SEM for five representative animals per group. *, < 0.05 compared with normal controls; , < 0.05 compared with AIA controls (Mann–Whitney test).<p><b>Copyright information:</b></p><p>Taken from "Anti-inflammatory effect of antidiabetic thiazolidinediones prevents bone resorption rather than cartilage changes in experimental polyarthritis"</p><p>Arthritis Research & Therapy 2008;10(1):R6-R6.</p><p>Published online 16 Jan 2008</p><p>PMCID:PMC2374462.</p><p></p

Effect of rosiglitazone and pioglitazone treatment on expression of PPAR target genes during adjuvant-induced arthritis

Animals were treated daily for 21 days with rosiglitazone (ROSI) 10 mg/kg or pioglitazone (PIO) 30 mg/kg by oral administration. Arthritic (adjuvant-induced arthritis (AIA)) and normal controls were given 0.5% carboxymethylcellulose alone. mRNA levels of adiponectin and peroxisome proliferator-activated receptor (PPAR)-γ normalized to RP29 in adipose tissue were assessed by RT-quantitative polymerase chain reaction. Data are expressed as means ± SEM for three representative samples per group (arthritis score close to the mean score of the group). , < 0.05 compared with AIA controls (ANOVA and Fisher's PLSD post-hoc test).<p><b>Copyright information:</b></p><p>Taken from "Anti-inflammatory effect of antidiabetic thiazolidinediones prevents bone resorption rather than cartilage changes in experimental polyarthritis"</p><p>Arthritis Research & Therapy 2008;10(1):R6-R6.</p><p>Published online 16 Jan 2008</p><p>PMCID:PMC2374462.</p><p></p

Effect of PPARγ overexpression on the inhibition of IL-1β-induced responses by 15d-PGJ

<p><b>Copyright information:</b></p><p>Taken from "Contrasting effects of peroxisome-proliferator-activated receptor (PPAR)γ agonists on membrane-associated prostaglandin Esynthase-1 in IL-1β-stimulated rat chondrocytes: evidence for PPARγ-independent inhibition by 15-deoxy-Δprostaglandin J"</p><p>Arthritis Research & Therapy 2005;7(6):R1325-R1337.</p><p>Published online 22 Sep 2005</p><p>PMCID:PMC1297580.</p><p>Copyright © 2005 Bianchi et al.; licensee BioMed Central Ltd.</p> Chondrocytes in six-well plates were transfected with pcDNA3.1 peroxisome-proliferator-activated receptor γ (PPARγ) construct (500 ng) for 36 hours. Thereafter, cells were pretreated for 4 hours with 10 μM 15-deoxy-Δprostaglandin J(15d-PGJ), then stimulated with 10 ng/ml IL-1β for 24 hours before extraction of total RNA and collection of culture supernatant. PGElevels assayed by ELISA in culture supernatant; relative abundance of microsomal prostaglandin E synthase-1 (mPGES-1) mRNAs analysed by real-time PCR and normalized to S29 mRNA; western blot control experiment of PPARγ and β-actin expression; modulation of adiponectin (a PPARγ target gene) mRNAs by PPARγ agonists and pcDNA3.1 PPARγ construct, analysed by real-time PCR and normalized to S29 mRNA. Results are expressed as means ± SD for at least three independent experiments. Statistically significant differences (< 0.05): *, comparison with non-stimulated controls; , comparison with IL-1β-stimulated cells; , comparison with PPARγ agonists alone or in combination with PPARγ plasmid

Effect of PPARγ agonists on IL-1β-induced prostaglandins levels, COX-2 and mPGES-1 mRNAs

<p><b>Copyright information:</b></p><p>Taken from "Contrasting effects of peroxisome-proliferator-activated receptor (PPAR)γ agonists on membrane-associated prostaglandin Esynthase-1 in IL-1β-stimulated rat chondrocytes: evidence for PPARγ-independent inhibition by 15-deoxy-Δprostaglandin J"</p><p>Arthritis Research & Therapy 2005;7(6):R1325-R1337.</p><p>Published online 22 Sep 2005</p><p>PMCID:PMC1297580.</p><p>Copyright © 2005 Bianchi et al.; licensee BioMed Central Ltd.</p> After 4 hours of pretreatment with 10 μM 15-deoxy-Δprostaglandin J(15d-PGJ) or rosiglitazone, chondrocytes were incubated with 10 ng/ml IL-1β for 12 or 24 hours. PGEand 6-keto-PGFlevels assayed by ELISA in culture supernatant; relative abundances of cyclo-oxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) mRNAs, analysed by real-time PCR and normalized to S29 mRNA COX-2 and mPGES-1 protein levels assessed by western blotting and normalized to β-actin level. Results are expressed as means ± SD for at least three independent experiments. Statistically significant differences (< 0.05): *, comparison with non-stimulated controls; , comparison with IL-1β-stimulated cells

Time course of prostaglandins production, COX-2 and mPGES-1 mRNA expression, in IL-1β-stimulated chondrocytes

<p><b>Copyright information:</b></p><p>Taken from "Contrasting effects of peroxisome-proliferator-activated receptor (PPAR)γ agonists on membrane-associated prostaglandin Esynthase-1 in IL-1β-stimulated rat chondrocytes: evidence for PPARγ-independent inhibition by 15-deoxy-Δprostaglandin J"</p><p>Arthritis Research & Therapy 2005;7(6):R1325-R1337.</p><p>Published online 22 Sep 2005</p><p>PMCID:PMC1297580.</p><p>Copyright © 2005 Bianchi et al.; licensee BioMed Central Ltd.</p> Rat cells were exposed to 10 ng/ml IL-1β for 6, 12, 24, 36 or 48 hours before total RNA extraction and collection of culture supernatant. Prostaglandin levels (PGE, 6-keto-PGF) assayed by ELISA in culture supernatant; relative abundances of cyclo-oxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) mRNAs, analysed by real-time PCR and normalized to S29 mRNA. Prostaglandin levels and PCR COX-2/S29 or mPGES-1/S29 mRNA ratios presented in histograms are expressed as means ± SD for at least three independent experiments. Statistically significant differences (< 0.05) from controls: * for PGEor COX-2; for 6-keto-PGFor mPGES-

Composition of fecal microbiota in this model.

<p>(A) Composition of fecal microbiota (Family level) of control, arthritic, colitic or arthritic and colitic mice (N = 5) at days 14 (before induction of colitis), 21 (during colitis), 30 (after colitis) and 41 (after colitis). (B) Differential analysis between colitis and colitis + arthritis groups at day 21. (C) Differential analysis between arthritis and colitis + arthritis groups at day 21.</p