recombinant human leptin rhleptin  (R&D Systems)

Journal: Arthritis Research & Therapy

Article Title: Local leptin production in osteoarthritis subchondral osteoblasts may be responsible for their abnormal phenotypic expression

doi: 10.1186/ar2925

Figure Lengend Snippet: Production of leptin in normal and OA osteoblasts . The expression of leptin was first determined by qPCR. Confluent osteoblasts (Ob) were lized in TRIzol and RNA extracted as described in Material and methods. RNA (1 μg) was reversed transcribed followed by qPCR amplification of 100 ng cDNA using specific primers for leptin and GAPDH. The data were processed with the GeneAmp 5700 SDS software and given as threshold cycle (Ct), corresponding to the PCR cycle at which an increase in reporter fluorescence above baseline signal can first be detected. The Ct was converted to the number of molecules and the values for each sample calculated as the ratio of the number of molecules of the target gene/number of molecules of GAPDH. A ) Quantification of leptin mRNA using Lep1 and Lep2 primers. Results are given as the mean value of markers relative to GAPDH ± SEM of n = 4 OA preparations. B ) Quantification of leptin mRNA levels in normal and OA Ob using Lep1 primers. Results are the mean ± SEM of n = 5 normal and n = 15 OA individual Ob preparations. C ) OA Ob were exposed to increasing doses of leptin and lepin mRNA levels were determined using Lep1 primers. Results are the mean ± SEM of n = 4 preparations. The protein production of leptin was next detected using a very selective ELISA. Conditioned-media of confluent normal and OA Ob incubated in HAM's F12/DMEM media containing 0.5% FBS for their last 48 hours of culture were recuperated and stored at -80°C. D ) Aliquots were taken to measure leptin using a very sensitive ELISA. Results are the mean ± SEM of n = 5 normal and n = 6 OA individual Ob preparations.

Article Snippet: For the determiniation of phenotypic markers, cells were either treated with 1 μg/ml recombinant human leptin (rhleptin, Calbiochem, San Diego, California, USA), 10 μg/ml recombinant human leptin R/Fc chimera (R&D Systems, Minneapolis, MN, USA) that neutralizes the activity of rhleptin, 100 μM Tyrphostin (AG490, Sigma-Aldrich), 75 μM piceatannol (Pce, Sigma-Aldrich), or the vehicle.

Techniques: Expressing, Amplification, Software, Fluorescence, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Arthritis Research & Therapy

Article Title: Local leptin production in osteoarthritis subchondral osteoblasts may be responsible for their abnormal phenotypic expression

doi: 10.1186/ar2925

Figure Lengend Snippet: Production of leptin receptors (OB-Rb) in normal and OA osteoblasts . The expression of leptin receptors was first determined by qPCR. A ) Confluent Ob were lized in TRIzol and RNA extracted as described in Material and Methods. RNA was reversed transcribed followed by PCR amplification of 100 ng cDNA as described in Figure 1 using OB-Rb and GAPDH primers. Results are the mean ± SEM of n = 7 normal and n = 19 OA Ob preparations, P < 0.004 vs normal and OA. B ) OA Ob were incubated for 24 hours with increasing concentrations of exogenous leptin. Cells were then lyzed and used for PCR amplification of OB-Rb as in A. Results are the mean ± SEM of n = 6 OA Ob preparations. Second, the production of leptin receptors was determined by Western blot analysis. C ) Confluent Ob were treated for 48 hours with or without 1,25(OH) 2 D 3 (50 nM), leptin (100 ng/ml), TGF-β1 (10 ng/ml) or HGF (10 ng/ml). The cells were then lized in RIPA buffer prior to separation using SDS-PAGE and Western blotting using specific antibodies to OB-Rb.

Article Snippet: For the determiniation of phenotypic markers, cells were either treated with 1 μg/ml recombinant human leptin (rhleptin, Calbiochem, San Diego, California, USA), 10 μg/ml recombinant human leptin R/Fc chimera (R&D Systems, Minneapolis, MN, USA) that neutralizes the activity of rhleptin, 100 μM Tyrphostin (AG490, Sigma-Aldrich), 75 μM piceatannol (Pce, Sigma-Aldrich), or the vehicle.

Techniques: Expressing, Amplification, Incubation, Western Blot, SDS Page

Journal: Arthritis Research & Therapy

Article Title: Local leptin production in osteoarthritis subchondral osteoblasts may be responsible for their abnormal phenotypic expression

doi: 10.1186/ar2925

Figure Lengend Snippet: Cellular proliferation and intracellular signaling of OA osteoblasts in response to leptin . OA osteoblasts were plated at 10,000 cells/cm 2 and allowed to attach overnight in HAM's F12/DMEM media containing 10% FBS. Cells were then treated with the same media with 0.5% FBS for 24 hours prior to receiving increasing doses of leptin (10 ng/ml, 100 ng/ml, 1 mg/ml or 10 mg/ml) or the vehicle in the same media for another incubation of 24 hours. Cell proliferation was assessed by the incorporation of BrdU or MTT assay. A ) Incorporation of BrdU by OA Ob in response to leptin; B ) Proliferation of OA Ob by MTT assay; C) Representative phospho p42/44 Western blot analysis in response to increasing doses of leptin in OA Ob. D ) Determination of phospho p42/44 levels using the NIH Image program developed at the U.S. National Institutes of Health with the Scion Image 1.63 program . E) Representative phospho p38 Western blot analysis in response to increasing doses of leptin in OA Ob. F) Determination of phospho p38 levels using the NIH Image program developed at the U.S. National Institutes of Health with the Scion Image 1.63 program . Values are the mean ± SEM of at least four separate experiments; * P < 0.05, ** P < 0.01.

Article Snippet: For the determiniation of phenotypic markers, cells were either treated with 1 μg/ml recombinant human leptin (rhleptin, Calbiochem, San Diego, California, USA), 10 μg/ml recombinant human leptin R/Fc chimera (R&D Systems, Minneapolis, MN, USA) that neutralizes the activity of rhleptin, 100 μM Tyrphostin (AG490, Sigma-Aldrich), 75 μM piceatannol (Pce, Sigma-Aldrich), or the vehicle.

Techniques: Incubation, MTT Assay, Western Blot

Journal: Arthritis Research & Therapy

Article Title: Local leptin production in osteoarthritis subchondral osteoblasts may be responsible for their abnormal phenotypic expression

doi: 10.1186/ar2925

Figure Lengend Snippet: Modulation of alkaline phosphatase, osteocalcin and collagen type 1 in OA Ob by inactivating leptin signaling . Confluent normal and OA Ob were treated for their last two days of culture with either media alone containing 0.5% FBS with or without 1,25(OH) 2 D 3 (50 nM) as per indicated for the individual markers. Cells were treated with either exogenous leptin, antibodies against leptin, tyrphostin (AG490, 100 μM) or Piceatannol (Pce, 75 μM) for 30 minutes prior to the addition of 1,25(OH) 2 D 3 except for CICP that was performed in the absence of 1,25(OH) 2 D 3 . At the end of the 48 h incubation, the supernatant was kept for osteocalcin and for collagen production, and cells were lyzed in ALPase buffer prior to measuring alkaline phosphatase activity by substrate hydrolysis. A ) Results of alkaline phosphatase activity for normal OB; B ) Results of alkaline phosphatase activity for OA OB; C ) Results of osteocalcin release by OA Ob; D ) Results of CICP production; E ) Confluent OA Ob were incubated in Ham's F12/DMEM media without serum and containing 1% ITS. Cells were treated with or without exogenous leptin, tyrphostin (AG490, 100 μM) or Piceatannol (Pce, 75 μM) for their last 48 hours of culture. Results of TGF-β1 levels in supernatants are shown. The results are the mean ± SEM of n = 4 normal and n = 9 OA Ob preparations.

Article Snippet: For the determiniation of phenotypic markers, cells were either treated with 1 μg/ml recombinant human leptin (rhleptin, Calbiochem, San Diego, California, USA), 10 μg/ml recombinant human leptin R/Fc chimera (R&D Systems, Minneapolis, MN, USA) that neutralizes the activity of rhleptin, 100 μM Tyrphostin (AG490, Sigma-Aldrich), 75 μM piceatannol (Pce, Sigma-Aldrich), or the vehicle.

Techniques: Incubation, Activity Assay

Journal: Arthritis Research & Therapy

Article Title: Local leptin production in osteoarthritis subchondral osteoblasts may be responsible for their abnormal phenotypic expression

doi: 10.1186/ar2925

Figure Lengend Snippet: Modulation of alkaline phosphatase and osteocalcin release in OA Ob by inactivating leptin or leptin signaling . OA Ob were treated with siRNA for either leptin or OB-Rb or a scrambled RNA as described in Material and methods. Cells were then used to determine alkaline phosphatase activity and osteocalcin release. A ) Results of alkaline phosphatase activity in response to leptin or OB-Rb siRNA treatments. B ) Results of osteocalcin release in response to leptin or OB-Rb siRNA treatments. C ) Leptin expression in response to siRNA. D ) OB-Rb expression in response to siRNA. Results are the mean ± SEM of n = 6 OA Ob preparations.

Article Snippet: For the determiniation of phenotypic markers, cells were either treated with 1 μg/ml recombinant human leptin (rhleptin, Calbiochem, San Diego, California, USA), 10 μg/ml recombinant human leptin R/Fc chimera (R&D Systems, Minneapolis, MN, USA) that neutralizes the activity of rhleptin, 100 μM Tyrphostin (AG490, Sigma-Aldrich), 75 μM piceatannol (Pce, Sigma-Aldrich), or the vehicle.

Techniques: Activity Assay, Expressing