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1.
Figure 1

Figure 1. IL-6 secretion is dose dependently inhibited by SB203580 in MC3T3-E1 cells.. From: p38 MAPK Regulates IL-1β Induced IL-6 Expression Through mRNA Stability in Osteoblasts.

MC3T3-E1 cells were treated with SB203580 at indicated concentrations for 30 minutes and then stimulated with IL-1β for 24 hours. Cell cultured supernatants were harvested and analyzed for IL-6 by ELISA. Data are presented as means of duplicate experiments measured in triplicate.

Chetan Patil, et al. Immunol Invest. ;33(2):213-233.
2.
Figure 2

Figure 2. p38 MAPK phosphorylation is dose-dependently inhibited by SB203580. From: p38 MAPK Regulates IL-1β Induced IL-6 Expression Through mRNA Stability in Osteoblasts.

MC3T3-E1 cells were serum starved, then treated with SB203580 at indicated concentrations for 30 minutes, and then stimulated for 20 minutes with IL-1β (1ng/ml). Whole cell extracts were analyzed by Western blot with antibodies against phosphorylated and non-phosphorylated p38 MAPK. Data is representative experiment preformed independently three times with similar results.

Chetan Patil, et al. Immunol Invest. ;33(2):213-233.
3.
Figure 3B

Figure 3B. De novo protein synthesis is required for p38 MAPK-mediated IL-6 mRNA synthesis in MC3T3-E1 Cells.. From: p38 MAPK Regulates IL-1β Induced IL-6 Expression Through mRNA Stability in Osteoblasts.

MC3T3-E1 cells with treated with IL-1 (1.0ng/ml) and SB203580 (2μM) and in the presence or absence of cycloheximide (CHX; 2μg/ml). Semi-quantitative RT-PCR was performed to evaluate IL-6 mRNA expression. See text for details on CHX effects. Means from two indepednent experiments are presented.

Chetan Patil, et al. Immunol Invest. ;33(2):213-233.
4.
Figure 5

Figure 5. p38 MAP kinase mediates IL-1β-induced IL-6 mRNA stability.. From: p38 MAPK Regulates IL-1β Induced IL-6 Expression Through mRNA Stability in Osteoblasts.

MC3T3-E1 cells were treated as in , and then actinomycin D (2μg/ml) was added for 30 and 60 minutes. Total RNA was harvested and analyzed by real time RT-PCR as above. Results are expressed as percentage of IL-6 mRNA remaining after normalized to GAPDH. IL-6 mRNA t½ was calculated to be >120 minutes with IL-1β treatment and reduced to ~24min with SB203580 (2μM). Means from two indepedent experiments are presnented.

Chetan Patil, et al. Immunol Invest. ;33(2):213-233.
5.
Figure 3A

Figure 3A. p38 MAP kinase is required for IL-1β-induced IL-6 gene expression.. From: p38 MAPK Regulates IL-1β Induced IL-6 Expression Through mRNA Stability in Osteoblasts.

MC3T3-E1 cells were treated with SB203580 (2μM), then stimulated with IL-1βfor 18 hours. Total RNA was isolated and real time RT-PCR performed for IL-6 and GAPDH using SybrGreen dye (ABI) on an iCycler PCR machine (BioRad). Threshold cycle values were used to quantitate normalized expression through QGene Software and expressed as ratio of target gene to reference gene (IL-6: GAPDH). Mean normalized expression ratios from 4 indepednent experiments are presented.

Chetan Patil, et al. Immunol Invest. ;33(2):213-233.
6.
Figure 6E

Figure 6E. Upstream signaling intermediates of p38 MAPK increase GFP expression in MCT-GFP-IL-6 3′UTR cells.. From: p38 MAPK Regulates IL-1β Induced IL-6 Expression Through mRNA Stability in Osteoblasts.

Cells were transduced with viral particles containing constitutively active MKK-6, MKK-3, or empty vector viral particles in the presence of polybream for 48 hours. GFP expression was measured by FACscan (10,000 cells were sorted in each group). Data indicates that both MKK-6 and MKK-3 can increase GFP expression in MCT-GFP-IL-6 3′UTR cells. These data suggest that both immediate upstream signaling intermediates of p38 MAPK are able to increase IL-6 mRNA reporter activity.

Chetan Patil, et al. Immunol Invest. ;33(2):213-233.
7.
Figure 6C

Figure 6C. p38 MAP kinase is required for IL-1β-induced IL-6 gene expression in MC6 cells.. From: p38 MAPK Regulates IL-1β Induced IL-6 Expression Through mRNA Stability in Osteoblasts.

MC6 cells were treated with SB203580 (2 or 5μM), then stimulated with IL-1β (1ng/ml) for 18 hours. Total RNA was isolated and real time RT-PCR performed for IL-6 and GAPDH using SybrGreen dye (ABI) on an iCycler PCR machine (BioRad). Threshold cycle values were used to quantitate normalized expression through QGene Software and expressed as ratio of target gene to reference gene (IL-6: GAPDH) compared to control untreated cells.

Chetan Patil, et al. Immunol Invest. ;33(2):213-233.
8.
Figure 6d

Figure 6d. p38 MAP kinase is required for IL-1β-induced eGFP gene expression in MC6 cells.. From: p38 MAPK Regulates IL-1β Induced IL-6 Expression Through mRNA Stability in Osteoblasts.

MC6 cells were treated with SB203580 (2 or 5μM), then stimulated with IL-1β (1ng/ml) for 18 hours. Total RNA was isolated and real time RT-PCR performed for eGFP and GAPDH using SybrGreen dye (ABI) on an iCycler PCR machine (BioRad). Threshold cycle values were used to quantitate normalized expression through Q-Gene Software and expressed as ratio of target gene to reference gene (eGFP: GAPDH) compared to control untreated cells.

Chetan Patil, et al. Immunol Invest. ;33(2):213-233.
9.
Figure 6B

Figure 6B. Various cytokines increase GFP expression of CMV-eGFP-IL-6 3′UTR construct in MC3T3-E1 cells.. From: p38 MAPK Regulates IL-1β Induced IL-6 Expression Through mRNA Stability in Osteoblasts.

MC3T3-E1CMV-eGFP-IL-63′UTR cells were treated with DMSO (control), PTH (10 nM), TNF-α (5ng/ml), E.Coli LPS (10μg/ml), or IL-1β (1 ng/ml) for 48 hours. Cells were harvested and visualized by flow cytometry on a FACScan. Histogram plots showing the entire GFP-expressing population are overlapped to indicate an increase in the GFP expressing cells when cells are stimulated with IL-1β or LPS. Lesser changes were observed with PTH or TNFα in this experiment. 10,000 cells were gated in this experiment for each treatment group.

Chetan Patil, et al. Immunol Invest. ;33(2):213-233.
10.
Figure 6A

Figure 6A. Diagram of the IL-6 gene structure and GFP-IL-6 3′UTR Reporter Construction.. From: p38 MAPK Regulates IL-1β Induced IL-6 Expression Through mRNA Stability in Osteoblasts.

Relevant cis elements (5′UTR, coding region and 3′UTR) discussed in proposal are indicated. As part of the preliminary data presented in this section, the murine IL-6 3′UTR (~390 bps) has been cloned into pEGFP-C1 vector where the SV40 poly adenylation site has been removed permitting expression of the EGFP gene under control of the CMV promoter. Stable clones of expressing this construct have been obtained in MC3T3-E1, NIH 3T3, and MC3T3-E1cells immortalized with SV40 large T antigen (designated MCT).

Chetan Patil, et al. Immunol Invest. ;33(2):213-233.
11.
Figure 4

Figure 4. p38 MAP kinase inhibition has minimal effect on proximal IL-6 promoter function.. From: p38 MAPK Regulates IL-1β Induced IL-6 Expression Through mRNA Stability in Osteoblasts.

MC3T3-E1 cells were transiently transfected with L-luciferase reporter constructs containing -225 and -138 bps of the IL-6 proximal promoter using Lipofectamide Plus (Invitrogen). Six hrs following transfection, cells were treated with SB203580 (5μM; Calbiochem) and stimulated with IL-1β (1ng/ml) for 24 hours. Cell extracts were normalized by cotransfection with an R-luciferase vector. Luminesence was measured using a Berthold Orion luminometer using the Dual luminescent kit (Promega) following manufacturers’ instructions. Representative data is presented. These experiments were performed twice independently with similar results.

Chetan Patil, et al. Immunol Invest. ;33(2):213-233.

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