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1.
Figure 8

Figure 8. From: Viral RNA Induces Type I Interferon-Dependent Cytokine Release and Cell Death in Mesangial Cells via Melanoma-Differentiation-Associated Gene-5 .

Intrarenal gene expression in nephrotoxic serum nephritis. Real-time RT-PCR was performed for RNA recognition receptors (A) and IFN-related genes (B) on total kidney RNA isolates from four mice of each group. Data are expressed as the ratio of the specific mRNA per respective 18S rRNA expression; P < 0.05 versus vehicle group.

Katharina Flür, et al. Am J Pathol. 2009 November;175(5):2014-2022.
2.
Figure 4

Figure 4. From: Viral RNA Induces Type I Interferon-Dependent Cytokine Release and Cell Death in Mesangial Cells via Melanoma-Differentiation-Associated Gene-5 .

IFN-α and -β enhance Il-6 release and RNA recognition in mesangial cells. A: Primary mesangial cells were stimulated with increasing doses of IFN-α-γ as indicated. Supernatants were harvested after 24 hours and analyzed by ELISA for Il-6. Data represent means ± SEM from three independent experiments. P < 0.05 versus medium. B: Mesangial cells were prestimulated with 1000 U/ml IFN-α, -β, or -γ for 24 hours. The prestimulated cells were then exposed to increasing doses of pI:C RNA as indicated. Supernatants were harvested after 24 hours and analyzed by ELISA for Il-6. Data represent means ± SEM; P < 0.05 versus medium.

Katharina Flür, et al. Am J Pathol. 2009 November;175(5):2014-2022.
3.
Figure 1

Figure 1. From: Viral RNA Induces Type I Interferon-Dependent Cytokine Release and Cell Death in Mesangial Cells via Melanoma-Differentiation-Associated Gene-5 .

Mesangial cells produce Il-6 and IFN-β on exposure to poly I:C RNA. Mesangial cells were prepared from C57BL/6 mice as described in Materials and Methods. Cultured mesangial cells were stimulated with increasing doses of poly I:C RNA, and Il-6 mRNA expression was determined by real-time RT-PCR after 1, 3, and 6 hours of stimulation with increasing doses of poly I:C RNA as indicated. Data are expressed as the ratio of Il-6 mRNA per respective 18S rRNA expression. Data are means ± SEM from three experiments, each analyzed in duplicate. P < 0.05 versus medium control.

Katharina Flür, et al. Am J Pathol. 2009 November;175(5):2014-2022.
4.
Figure 5

Figure 5. From: Viral RNA Induces Type I Interferon-Dependent Cytokine Release and Cell Death in Mesangial Cells via Melanoma-Differentiation-Associated Gene-5 .

Poly I:C (pI:C) RNA-induced Il-6 release in mesangial cells depends on type I IFNs. A: Primary mesangial cells were stimulated with increasing doses of pI:C RNA/cationic lipid (CL) complexes as indicated. At given concentrations of such complexes, increasing doses of neutralizing antibodies against IFN-α and IFN-β were added. B: Mesangial cells were prepared from wild-type or IFNa-R−/− mice, and cells were stimulated as before. In A and B, supernatants were harvested after 24 hours and analyzed by ELISA for Il-6. Data are means ± SEM from four experiments each analyzed in duplicate. P < 0.05 versus no IFN-α and IFN-β antibody group.

Katharina Flür, et al. Am J Pathol. 2009 November;175(5):2014-2022.
5.
Figure 7

Figure 7. From: Viral RNA Induces Type I Interferon-Dependent Cytokine Release and Cell Death in Mesangial Cells via Melanoma-Differentiation-Associated Gene-5 .

Nephrotoxic serum nephritis in C57BL/6 mice. A: Urinary albumin/creatinine ratios were determined from all groups of mice at days 7 and 10 as described in Materials and Methods. Data represent means ± SEM; P < 0.05 versus day 0, ∗∗P < 0.05 versus vehicle on day 10. B: Focal glomerular necrosis was quantified on periodic acid-Schiff (PAS)-stained renal sections from all groups on day 10 applying a semiquantitative score from 0 to 3 in 15 glomeruli per section. Data represent means ± SEM; ∗∗P < 0.05 pI:C RNA/cationic lipid versus pI:C; P < 0.05 versus vehicle. C: Renal sections from mice of all groups were stained with PAS. Representative images were taken at an original magnification of ×100 and ×400. Focal necrosis is indicated by asterisk.

Katharina Flür, et al. Am J Pathol. 2009 November;175(5):2014-2022.
6.
Figure 2

Figure 2. From: Viral RNA Induces Type I Interferon-Dependent Cytokine Release and Cell Death in Mesangial Cells via Melanoma-Differentiation-Associated Gene-5 .

Viral dsRNA complexed with cationic lipids activates mesangial cells to produce high amounts of Il-6 and type I IFN via a Trif-independent pathway. Primary mesangial cells were isolated from wild-type C56BL/6 mice and Trif-mutant mice as described in Materials and Methods. Cells were stimulated with increasing doses of poly I:C (pI:C) RNA alone or complexed with the cationic lipid (CL) Lipofectamine. Supernatants were harvested after 24 hours and analyzed by ELISA for the following mediators: Il-6 (A), IFN-α (B), and IFN-β (C). Data are means ± SEM from three experiments each analyzed in duplicate and presented in a logarithmic scale. P < 0.05 wild-type versus medium; ∗∗P < 0.05 Trif-mutant versus wild type.

Katharina Flür, et al. Am J Pathol. 2009 November;175(5):2014-2022.
7.
Figure 6

Figure 6. From: Viral RNA Induces Type I Interferon-Dependent Cytokine Release and Cell Death in Mesangial Cells via Melanoma-Differentiation-Associated Gene-5 .

Cytosolic recognition of viral dsRNA-induced mesangial cells death. A: Primary mesangial cells were stimulated with increasing doses of poly I:C (pI:C) RNA with or without cationic lipid (CL) as indicated. After 72 hours, the number of proliferating cells was determined by a bioluminescence assay as described in Materials and Methods. P < 0.05 versus medium; ∗∗P < 0.05 versus sine CL. B: Primary mesangial cells were stimulated with poly I:C RNA+CL (pIC) in the presence or absence of the caspase-2 inhibitor Z-VDVAD-FMK (CI, 5 μg/ml), the caspase-8 inhibitor Ac-IETD-CHO (CI, 5 μg/ml), the caspase-9 inhibitor Z-LEHD-FMK (CI, 5 μg/ml), and the caspase-10 inhibitor Z-AEVD-FMK (CI, 5 μg/ml). After 72 hours, the number of proliferating cells was determined by a bioluminescence assay. P < 0.05 versus pI:C RNA. Note that all caspase inhibitors prevented pI:C RNA+CL-induced inhibition of cell growth. C: In similar experiments, mesangial cells from IFNa-R+/+ (black bars) and IFNa-R−/− cells (white bars) were stimulated with pI:C/cationic lipid complexes. P < 0.05 versus IFNa-R+/+.

Katharina Flür, et al. Am J Pathol. 2009 November;175(5):2014-2022.
8.
Figure 3

Figure 3. From: Viral RNA Induces Type I Interferon-Dependent Cytokine Release and Cell Death in Mesangial Cells via Melanoma-Differentiation-Associated Gene-5 .

Cytosolic RNA receptors in mesangial cells. A: Primary mesangial cells were cultured in the presence or absence of 2000 U/ml IFN-β (A) or increasing doses of rmIl-6 (B). After 6 hours, mRNA was harvested, and real-time RT-PCR was performed for various RNA recognition molecules, as indicated. Data are expressed as the ratio of the specific mRNA per respective 18S rRNA expression. n.d., not detected; P < 0.05 versus medium. C: Primary mesangial cells were cultured in the presence or absence of poly I:C (pI:C) RNA/CL complexes over various time periods, as indicated. Unstimulated J774 macrophages served as a positive control. Protein expression of Tlr3, Rig-I, and Mda5 was determined by Western blot. β-Actin expression was used as a loading control. D and E: Mesangial cells were transfected twice with Mda-5-specific siRNA (D), Rig-I-specific siRNA (E), or nonspecific control RNA as described in Materials and Methods. Knockdown efficacy was tested by real-time PCR for the respective target (left panels). Impact of knockdown on pI:C recognition was determined by real-time PCR for Il-6 or Cxcl10 after 6 hours of incubation with 3 μg of pI:C RNA either complexed with cationic lipid (CL) or uncomplexed. Data represent means from three experiments ± SEM; P < 0.05 versus nonspecific control RNA (n.c.).

Katharina Flür, et al. Am J Pathol. 2009 November;175(5):2014-2022.

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