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NF-κB Activation Represses Tumor Necrosis Factor-α-induced Autophagy

Djavaheri-Mergny, Mojgan, Amelotti, Manuela, Mathieu, Julie, Besançon, Françoise, Bauvy, Chantal, Souquère, Sylvie, Pierron, Gérard, Codogno, Patrice·2006·Journal of Biological Chemistry

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Crossref · 10.1074/jbc.m602097200

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FIGURE2 TheeffectofTNF 𝛂 onlonglivedproteindegradationinEW7PCandEW7MAD1cells. Therateof proteolysis of [ 14 C]valine-labeled long lived proteins was measured in cells following an 8-h incubation with TNF 𝛂 in the presence and absence of 10 mM 3-MA. As a positive control, cells were subjected to 4-h starvation in the presence and absence of 10 mM 3-MA. Results are the mean ± S.D. of three independent experiments.
FIGURE2 TheeffectofTNF 𝛂 onlonglivedproteindegradationinEW7PCandEW7MAD1cells. Therateof proteolysis of [ 14 C]valine-labeled long lived proteins was measured in cells following an 8-h incubation with TNF 𝛂 in the presence and absence of 10 mM 3-MA. As a positive control, cells were subjected to 4-h starvation in the presence and absence of 10 mM 3-MA. Results are the mean ± S.D. of three independent experiments.CC-BY · From the PDF
FIGURE 3 Inactivation of NF𝛋 B in NB4 and MCF7 cells induces autophagy in response to TNF 𝛂 . NF𝛋 Bcompetent NB4 cells (NB4/GFP), NB4 cells expressing an inhibitor of NF𝛋 B activation (NB4/GFP-MAD), MCF7 cells, and MCF7 cells transiently transfected with siRNA against p65 were treated with TNF 𝛂 (2000 units/ml) for different times as indicated. A and D , representative control cells and TNF 𝛂 -treated cells labeled with MDC are shown. Bar , 3 𝛍 m. B and E , the percentage of cells with MDC-stained dots was quantified after treatment with TNF 𝛂 of NB4 cells or MCF7 cells, for 8 or 32 h, respectively ( black bar ). Inset , the levels of p65 protein expression in the presence and absence of p65 siRNA are shown. For each condition, cells were also subjected to Hoechst staining, and the percentage of apoptotic nuclei was scored ( white bar ). C and F , immunoblot analysis of LC3-I processing into LC3-II in NB4/GFP and NB4/GFPMAD cells treated in the presence and absence of TNF 𝛂 ( C ) and in MCF7 cells incubated for 32 h with siRNA against p65 before the addition of TNF 𝛂 ( F ).
FIGURE 3 Inactivation of NF𝛋 B in NB4 and MCF7 cells induces autophagy in response to TNF 𝛂 . NF𝛋 Bcompetent NB4 cells (NB4/GFP), NB4 cells expressing an inhibitor of NF𝛋 B activation (NB4/GFP-MAD), MCF7 cells, and MCF7 cells transiently transfected with siRNA against p65 were treated with TNF 𝛂 (2000 units/ml) for different times as indicated. A and D , representative control cells and TNF 𝛂 -treated cells labeled with MDC are shown. Bar , 3 𝛍 m. B and E , the percentage of cells with MDC-stained dots was quantified after treatment with TNF 𝛂 of NB4 cells or MCF7 cells, for 8 or 32 h, respectively ( black bar ). Inset , the levels of p65 protein expression in the presence and absence of p65 siRNA are shown. For each condition, cells were also subjected to Hoechst staining, and the percentage of apoptotic nuclei was scored ( white bar ). C and F , immunoblot analysis of LC3-I processing into LC3-II in NB4/GFP and NB4/GFPMAD cells treated in the presence and absence of TNF 𝛂 ( C ) and in MCF7 cells incubated for 32 h with siRNA against p65 before the addition of TNF 𝛂 ( F ).CC-BY · From the PDF
FIGURE 4 ROS scavengers inhibit TNF 𝛂 -induced autophagy in EW7MAD1 cells. EW7MAD1 and EW7PC cells were treated with either BHA (100 𝛍 M) or BNP (50 𝛍 M) for 2 h prior to treatment with TNF 𝛂 (2000 units/ml, 8 h) and H 2 O2 (100 𝛍 M, 8 h), respectively. A , fluorescence microscopy was performed following MDC staining. Representative images of cells with MDC staining are shown. Bar , 6 𝛍 m. B , EW7PC and EW7MAD1 cells were transfected with GFP-LC3 plasmids and then subjected to treatment as indicated in A . For each condition, the percentageofcells with punctate GFP-LC3 staining was quantified. Results shown in A and B are representative of three independent experiments.
FIGURE 4 ROS scavengers inhibit TNF 𝛂 -induced autophagy in EW7MAD1 cells. EW7MAD1 and EW7PC cells were treated with either BHA (100 𝛍 M) or BNP (50 𝛍 M) for 2 h prior to treatment with TNF 𝛂 (2000 units/ml, 8 h) and H 2 O2 (100 𝛍 M, 8 h), respectively. A , fluorescence microscopy was performed following MDC staining. Representative images of cells with MDC staining are shown. Bar , 6 𝛍 m. B , EW7PC and EW7MAD1 cells were transfected with GFP-LC3 plasmids and then subjected to treatment as indicated in A . For each condition, the percentageofcells with punctate GFP-LC3 staining was quantified. Results shown in A and B are representative of three independent experiments.CC-BY · From the PDF
FIGURE 5 Comparison of autophagy-regulated protein expression in TNF 𝛂 -treated EW7PC cells and in TNF 𝛂 -treated EW7MAD cells. A , EW7PC and EW7MAD1 cells were treated for the indicated times with TNF 𝛂 before cell lysis. Equal protein amounts of whole cell extracts were analyzed by Western blotting using antibodies directed against phospho-4E-BP1, 4E-BP1, phospho-p70 S6 kinase, p70 S6 kinase, Beclin 1, Atg7 and Atg5, Bcl-2, and actin. B , EW7PC and EW7MAD1cells were treated for the indicated times with either TNF 𝛂 (2000 units/ml) or H 2 O2 (100 𝛍 M). Left , cellular extracts were prepared and analyzed for the expression level of Beclin 1. The bands corresponding to the Beclin 1 protein level in EW7MAD1 cells were quantified using NIH Image software and were normalized to actin expression level ( right ). Right , total RNA was extracted from TNF 𝛂 -treated EW7MAD1 cells before the measurement of the Beclin 1 mRNA level by real time quantitative reverse transcription-PCR. Values obtained for Beclin 1 mRNA transcripts were normalized to those of 𝛃 -actin. C , EW7MAD1 and EW7PC cells were first treated with BHA (100 𝛍 M) and then incubated with TNF 𝛂 (2000 units/ml, 1 h) or H2O2 (100 𝛍 M, 0.5 h), respectively. Cellular extracts were prepared and analyzed for Beclin 1 expression by Western blotting. Results shown in A-C are representative of three independent experiments.
FIGURE 5 Comparison of autophagy-regulated protein expression in TNF 𝛂 -treated EW7PC cells and in TNF 𝛂 -treated EW7MAD cells. A , EW7PC and EW7MAD1 cells were treated for the indicated times with TNF 𝛂 before cell lysis. Equal protein amounts of whole cell extracts were analyzed by Western blotting using antibodies directed against phospho-4E-BP1, 4E-BP1, phospho-p70 S6 kinase, p70 S6 kinase, Beclin 1, Atg7 and Atg5, Bcl-2, and actin. B , EW7PC and EW7MAD1cells were treated for the indicated times with either TNF 𝛂 (2000 units/ml) or H 2 O2 (100 𝛍 M). Left , cellular extracts were prepared and analyzed for the expression level of Beclin 1. The bands corresponding to the Beclin 1 protein level in EW7MAD1 cells were quantified using NIH Image software and were normalized to actin expression level ( right ). Right , total RNA was extracted from TNF 𝛂 -treated EW7MAD1 cells before the measurement of the Beclin 1 mRNA level by real time quantitative reverse transcription-PCR. Values obtained for Beclin 1 mRNA transcripts were normalized to those of 𝛃 -actin. C , EW7MAD1 and EW7PC cells were first treated with BHA (100 𝛍 M) and then incubated with TNF 𝛂 (2000 units/ml, 1 h) or H2O2 (100 𝛍 M, 0.5 h), respectively. Cellular extracts were prepared and analyzed for Beclin 1 expression by Western blotting. Results shown in A-C are representative of three independent experiments.CC-BY · From the PDF
Image 6
CC-BY · From the PDF
FIGURE 7 Effect of caspase inhibition on TNF 𝛂 -induced autophagy in EW7MAD1cells. EW7MAD1cellswerefirstincubatedfor2hwithZ-VAD-fmk (50 𝛍 M) and then treated for 8 h with TNF 𝛂 The bar represents 6 𝛍 m. A , cells were subjected to MDC staining before analysis by fluorescence microscopy. B , cellular extracts were prepared for Western blotting analysis of LC3-II expression. Results shown in A and B are representative of three independent experiments.
FIGURE 7 Effect of caspase inhibition on TNF 𝛂 -induced autophagy in EW7MAD1cells. EW7MAD1cellswerefirstincubatedfor2hwithZ-VAD-fmk (50 𝛍 M) and then treated for 8 h with TNF 𝛂 The bar represents 6 𝛍 m. A , cells were subjected to MDC staining before analysis by fluorescence microscopy. B , cellular extracts were prepared for Western blotting analysis of LC3-II expression. Results shown in A and B are representative of three independent experiments.CC-BY · From the PDF

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Metadata

Authors
Djavaheri-Mergny, M., Amelotti, M., Mathieu, J., Besançon, F., Bauvy, C., Souquère, S., Pierron, G., & Codogno, P.
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2006-10
Volume
281
Issue
41
Pages
30373-30382
ISSN
0021-9258
Cited by
407
References
5
Language
en

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