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pdonr223 eef2k (Addgene inc)

Name: pdonr223 eef2k
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Addgene inc pdonr223 eef2k
Pdonr223 Eef2k, supplied by Addgene inc, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pdonr223 eef2k - by Bioz Stars, 2026-02

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Figure 1. Silencing of EEF2K induces autophagy in human colon cancer cells. ( A and B ) HT-29 or HCT-116 cells were transfected with nontargeting control siRNA (siCTL), a single siRNA duplex targeting EEF2K (si EEF2K ; A ) or multiple siRNAs targeting different regions of EEF2K (si EEF2K ; B ) for 48 h. EEF2K, phospho-EEF2 (Thr56; p-EEF2), EEF2, LC3, and ACTB/β-actin were analyzed by western blot. Representative western blot and densitometric analysis normalized to ACTB demonstrating the effect of EEF2K silencing on LC3-II levels. ( C and D ) HT-29 or HCT-116 cells were transfected with control siRNA or a single siRNA duplex targeting EEF2K for 48 h. ( C ) Representative immunofluorescent images showing redistribution of autophagic marker LC3 in EEF2K knockdown cells were taken on a confocal microscope. Cells were fixed with 3.5% formaldehyde for 10 min, permeabilized with 0.1% Triton X-100 for 10 min, and stained with LC3 antibody and DAPI. Scale bar: 10 μm. ( D ) The average number of LC3 dots per cell was counted in more than 5 fields with at least 100 cells for each group. ( E ) Representative western blot and densitometric analysis normalized to ACTB demonstrating the effect of lysosomal protease inhibitors E64d plus pepstatin A on EEF2K silencing induced LC3-II accumulation. HT-29 cells were transfected with nontargeting control siRNA or EEF2K siRNA. At 3 h after transfection, cells were treated with 10 μg/ml E64d and pepstatin A (Pep A) for 45 h. All quantitative data shown represent the means ± SEM of at least 3 independent experiments. * P < 0.05, $ P < 0.01, and # P < 0.001, vs. the siCTL group ( A, B, and D ) or vehicle treatment only ( E ).

Journal: Autophagy

Article Title: Silencing of EEF2K (eukaryotic elongation factor-2 kinase) reveals AMPK-ULK1-dependent autophagy in colon cancer cells

doi: 10.4161/auto.29164

Figure Lengend Snippet: Figure 1. Silencing of EEF2K induces autophagy in human colon cancer cells. ( A and B ) HT-29 or HCT-116 cells were transfected with nontargeting control siRNA (siCTL), a single siRNA duplex targeting EEF2K (si EEF2K ; A ) or multiple siRNAs targeting different regions of EEF2K (si EEF2K ; B ) for 48 h. EEF2K, phospho-EEF2 (Thr56; p-EEF2), EEF2, LC3, and ACTB/β-actin were analyzed by western blot. Representative western blot and densitometric analysis normalized to ACTB demonstrating the effect of EEF2K silencing on LC3-II levels. ( C and D ) HT-29 or HCT-116 cells were transfected with control siRNA or a single siRNA duplex targeting EEF2K for 48 h. ( C ) Representative immunofluorescent images showing redistribution of autophagic marker LC3 in EEF2K knockdown cells were taken on a confocal microscope. Cells were fixed with 3.5% formaldehyde for 10 min, permeabilized with 0.1% Triton X-100 for 10 min, and stained with LC3 antibody and DAPI. Scale bar: 10 μm. ( D ) The average number of LC3 dots per cell was counted in more than 5 fields with at least 100 cells for each group. ( E ) Representative western blot and densitometric analysis normalized to ACTB demonstrating the effect of lysosomal protease inhibitors E64d plus pepstatin A on EEF2K silencing induced LC3-II accumulation. HT-29 cells were transfected with nontargeting control siRNA or EEF2K siRNA. At 3 h after transfection, cells were treated with 10 μg/ml E64d and pepstatin A (Pep A) for 45 h. All quantitative data shown represent the means ± SEM of at least 3 independent experiments. * P < 0.05, $ P < 0.01, and # P < 0.001, vs. the siCTL group ( A, B, and D ) or vehicle treatment only ( E ).

Article Snippet: A plasmid pDONR223-EEF2K containing full-length of human EEF2K coding region was obtained from Addgene (Addgene plasmid 23726, USA).

Techniques: Transfection, Control, Western Blot, Marker, Knockdown, Microscopy, Staining

Figure 2. BECN1 and ATG7 are required for autophagy in response to EEF2K silencing. ( A ) Silencing of EEF2K upregulates the protein levels of BECN1 and ATG7, but not ATG5. HT-29 or HCT-116 cells were transfected with nontargeting control siRNA (siCTL) or a single EEF2K siRNA duplex (si EEF2K ) for 48 h. ATG5, BECN1, ATG7, and ACTB were analyzed by western blot. Data shown are representative of more than 3 independent experiments. ( B ) Silencing of EEF2K does not change the mRNA levels of ATG5 , BECN1, and ATG7 . Cells were transfected as in ( A ). The mRNA levels of ATG5 , BECN1, and ATG7 were analyzed by RT-PCR. ( C ) Effect of MG132 on the protein levels of ATG5, BECN1, and ATG7 in EEF2K knockdown cells. HCT-116 cells were transfected with nontargeting control siRNA (siCTL) or a single EEF2K siRNA duplex (si EEF2K ) for 48. Before harvested for western blot, cells were treated with MG132 (10 μM) for 12 h. ( D and E ) Representative western blot and densitometric analysis normalized to ACTB demonstrating the effects of BECN1 siRNA ( D ) and ATG7 siRNA ( E ) on LC3-II levels induced by EEF2K silencing. HT-29 cells were transfected with nontargeting siRNA, si EEF2K , BECN1 siRNA (si BECN1 ), ATG7 siRNA (si ATG7 ), si EEF2K plus si BECN1 , or si EEF2K plus si ATG7 for 48 h. All quantitative data shown represent the means ± SEM of at least 3 independent experiments. * P < 0.05 and $ P < 0.01, vs. the si EEF2K group. ( F ) The effects of BECN1 siRNA and ATG7 siRNA on LC3 dots accumulation induced by EEF2K silencing. HT-29 cells were treated with siRNAs against EEF2K , BECN1 , or ATG7 as in ( D and E ). Cells were fixed, stained for LC3, and imaged. The average number of LC3 dots per cell was counted in more than 5 fields with at least 100 cells for each group and expressed as the means ± SEM of 3 independent experiments. # P < 0.001, vs. the EEF2K siRNA group (si EEF2K ).

Journal: Autophagy

Article Title: Silencing of EEF2K (eukaryotic elongation factor-2 kinase) reveals AMPK-ULK1-dependent autophagy in colon cancer cells

doi: 10.4161/auto.29164

Figure Lengend Snippet: Figure 2. BECN1 and ATG7 are required for autophagy in response to EEF2K silencing. ( A ) Silencing of EEF2K upregulates the protein levels of BECN1 and ATG7, but not ATG5. HT-29 or HCT-116 cells were transfected with nontargeting control siRNA (siCTL) or a single EEF2K siRNA duplex (si EEF2K ) for 48 h. ATG5, BECN1, ATG7, and ACTB were analyzed by western blot. Data shown are representative of more than 3 independent experiments. ( B ) Silencing of EEF2K does not change the mRNA levels of ATG5 , BECN1, and ATG7 . Cells were transfected as in ( A ). The mRNA levels of ATG5 , BECN1, and ATG7 were analyzed by RT-PCR. ( C ) Effect of MG132 on the protein levels of ATG5, BECN1, and ATG7 in EEF2K knockdown cells. HCT-116 cells were transfected with nontargeting control siRNA (siCTL) or a single EEF2K siRNA duplex (si EEF2K ) for 48. Before harvested for western blot, cells were treated with MG132 (10 μM) for 12 h. ( D and E ) Representative western blot and densitometric analysis normalized to ACTB demonstrating the effects of BECN1 siRNA ( D ) and ATG7 siRNA ( E ) on LC3-II levels induced by EEF2K silencing. HT-29 cells were transfected with nontargeting siRNA, si EEF2K , BECN1 siRNA (si BECN1 ), ATG7 siRNA (si ATG7 ), si EEF2K plus si BECN1 , or si EEF2K plus si ATG7 for 48 h. All quantitative data shown represent the means ± SEM of at least 3 independent experiments. * P < 0.05 and $ P < 0.01, vs. the si EEF2K group. ( F ) The effects of BECN1 siRNA and ATG7 siRNA on LC3 dots accumulation induced by EEF2K silencing. HT-29 cells were treated with siRNAs against EEF2K , BECN1 , or ATG7 as in ( D and E ). Cells were fixed, stained for LC3, and imaged. The average number of LC3 dots per cell was counted in more than 5 fields with at least 100 cells for each group and expressed as the means ± SEM of 3 independent experiments. # P < 0.001, vs. the EEF2K siRNA group (si EEF2K ).

Article Snippet: A plasmid pDONR223-EEF2K containing full-length of human EEF2K coding region was obtained from Addgene (Addgene plasmid 23726, USA).

Techniques: Transfection, Control, Western Blot, Reverse Transcription Polymerase Chain Reaction, Knockdown, Staining

Figure 3. EEF2K silencing promotes cell survival in human colon cancer cells. ( A ) Representative western blot demonstrating the knockdown efficiency of EEF2K siRNA and the overexpression efficiency of EEF2K in both HT-29 and HCT-116 cells. Cells were transfected with control siRNA (siCTL), EEF2K siRNA (si EEF2K ), empty vector (Vector), or EEF2K plasmids (EEF2K) for 48 h. ( B and C ) The effects of EEF2K knockdown and EEF2K overexpression on cell viability. HT-29 or HCT-116 cells were transfected as in ( A ), and then assessed by the MTT assay. ( D and E ) The effects of EEF2K knockdown and EEF2K overexpression on colony formation. HT-29 or HCT-116 cells were transfected as in ( A ). After 48 h transfection, cells were seeded into 6-well plates at the density of 150 cells per well for control siRNA and EEF2K siRNA groups ( D ) and 200 cells per well for the empty vector and EEF2K overexpression groups ( E ), incubated at 37 °C for 12 to 14 d, stained with crystal violet (0.5% w/v) and imaged. Colonies with 50 or more cells were counted. ( F and G ) The effects of EEF2K knockdown and EEF2K overexpression on cell size. HT-29 or HCT-116 cells were transfected as in ( A ). After 48 h transfection, cells were imaged using a Nikon fluorescence microscope. Scale bar: 20 μm. Cell size was analyzed using the MetaMorph software. The amounts of cell size in more than 50 cells for each group were quantified. ( H and I ) The effects of EEF2K knockdown and EEF2K overexpression on cell number. HT-29 or HCT-116 cells were transfected as in ( A ). Cell number was quantified after 48 h transfection. ( J ) The effect of EEF2K siRNA on oxaliplatin induced apoptosis. HCT-116 cells were transfected with control siRNA or EEF2K siRNA for 24 h, and then treated with vehicle (0.1% DMSO) or oxaliplatin (25 μM) for 48 h. Cells were stained with ANXA5-PI. The percentage of apoptotic cells (ANXA5+) was analyzed by flow cytometry. ( K ) The effect of EEF2K overexpression on oxaliplatin-induced apoptosis. HT-29 cells were transfected with empty vector (Vector), or EEF2K plasmids (EEF2K) for 24 h, and then treated with vehicle (0.1% DMSO) or oxaliplatin (25 μM) for 48 h. Cells were stained with ANXA5-PI and analyzed by flow cytometry as in ( J ). All quantitative data shown represent the means ± SEM of at least 3 independent experiments. * P < 0.05, $ P < 0.01 and # P < 0.001, vs. the siCTL group ( B, D, F, and H ), the vector group ( C, E, G, and I ), or the oxaliplatin treatment only ( J and K ).

Journal: Autophagy

Article Title: Silencing of EEF2K (eukaryotic elongation factor-2 kinase) reveals AMPK-ULK1-dependent autophagy in colon cancer cells

doi: 10.4161/auto.29164

Figure Lengend Snippet: Figure 3. EEF2K silencing promotes cell survival in human colon cancer cells. ( A ) Representative western blot demonstrating the knockdown efficiency of EEF2K siRNA and the overexpression efficiency of EEF2K in both HT-29 and HCT-116 cells. Cells were transfected with control siRNA (siCTL), EEF2K siRNA (si EEF2K ), empty vector (Vector), or EEF2K plasmids (EEF2K) for 48 h. ( B and C ) The effects of EEF2K knockdown and EEF2K overexpression on cell viability. HT-29 or HCT-116 cells were transfected as in ( A ), and then assessed by the MTT assay. ( D and E ) The effects of EEF2K knockdown and EEF2K overexpression on colony formation. HT-29 or HCT-116 cells were transfected as in ( A ). After 48 h transfection, cells were seeded into 6-well plates at the density of 150 cells per well for control siRNA and EEF2K siRNA groups ( D ) and 200 cells per well for the empty vector and EEF2K overexpression groups ( E ), incubated at 37 °C for 12 to 14 d, stained with crystal violet (0.5% w/v) and imaged. Colonies with 50 or more cells were counted. ( F and G ) The effects of EEF2K knockdown and EEF2K overexpression on cell size. HT-29 or HCT-116 cells were transfected as in ( A ). After 48 h transfection, cells were imaged using a Nikon fluorescence microscope. Scale bar: 20 μm. Cell size was analyzed using the MetaMorph software. The amounts of cell size in more than 50 cells for each group were quantified. ( H and I ) The effects of EEF2K knockdown and EEF2K overexpression on cell number. HT-29 or HCT-116 cells were transfected as in ( A ). Cell number was quantified after 48 h transfection. ( J ) The effect of EEF2K siRNA on oxaliplatin induced apoptosis. HCT-116 cells were transfected with control siRNA or EEF2K siRNA for 24 h, and then treated with vehicle (0.1% DMSO) or oxaliplatin (25 μM) for 48 h. Cells were stained with ANXA5-PI. The percentage of apoptotic cells (ANXA5+) was analyzed by flow cytometry. ( K ) The effect of EEF2K overexpression on oxaliplatin-induced apoptosis. HT-29 cells were transfected with empty vector (Vector), or EEF2K plasmids (EEF2K) for 24 h, and then treated with vehicle (0.1% DMSO) or oxaliplatin (25 μM) for 48 h. Cells were stained with ANXA5-PI and analyzed by flow cytometry as in ( J ). All quantitative data shown represent the means ± SEM of at least 3 independent experiments. * P < 0.05, $ P < 0.01 and # P < 0.001, vs. the siCTL group ( B, D, F, and H ), the vector group ( C, E, G, and I ), or the oxaliplatin treatment only ( J and K ).

Article Snippet: A plasmid pDONR223-EEF2K containing full-length of human EEF2K coding region was obtained from Addgene (Addgene plasmid 23726, USA).

Techniques: Western Blot, Knockdown, Over Expression, Transfection, Control, Plasmid Preparation, MTT Assay, Incubation, Staining, Fluorescence, Microscopy, Software, Flow Cytometry

Figure 4. Cell survival induced by EEF2K silencing is attributed to induction of autophagy. ( A ) The effects of BECN1 siRNA and ATG7 siRNA on the increase of cell viability induced by EEF2K silencing. HT-29 or HCT-116 cells were transfected with BECN1 siRNA (si BECN1 ), ATG7 siRNA (si ATG7 ), EEF2K siRNA (si EEF2K ), si BECN1 plus si EEF2K , or si ATG7 plus si EEF2K for 48 h. Cell viability was assessed by the MTT assay. ( B ) The effects of BECN1 siRNA and ATG7 siRNA on the increase of colony formation induced by EEF2K silencing. HT-29 or HCT-116 cells were transfected with si BECN1 , si ATG7 , si EEF2K , si BECN1 plus si EEF2K , or si ATG7 plus si EEF2K . After 48 h transfection, cells were seeded into 6-well plates at the density of 150 cells per well, incubated at 37 °C for 12 to 14 d and then stained with crystal violet (0.5% w/v). Colonies with 50 or more cells were counted. ( C ) The effects of E64d and pepstatin A on cell viability in EEF2K silenced cells. HT-29 or HCT-116 cells were transfected with nontargeting control siRNA or EEF2K siRNA. After 3 h transfection, cells were treated with vehicle (0.1% DMSO), or 10 μg/ml E64d and pepstatin A for 45 h and then assessed by the MTT assay. All data shown are expressed as the means ± SEM of 3 independent experiments. * P < 0.05, $ P < 0.01, and # P < 0.001, vs. the EEF2K siRNA group (si EEF2K ; A–C ).

Journal: Autophagy

Article Title: Silencing of EEF2K (eukaryotic elongation factor-2 kinase) reveals AMPK-ULK1-dependent autophagy in colon cancer cells

doi: 10.4161/auto.29164

Figure Lengend Snippet: Figure 4. Cell survival induced by EEF2K silencing is attributed to induction of autophagy. ( A ) The effects of BECN1 siRNA and ATG7 siRNA on the increase of cell viability induced by EEF2K silencing. HT-29 or HCT-116 cells were transfected with BECN1 siRNA (si BECN1 ), ATG7 siRNA (si ATG7 ), EEF2K siRNA (si EEF2K ), si BECN1 plus si EEF2K , or si ATG7 plus si EEF2K for 48 h. Cell viability was assessed by the MTT assay. ( B ) The effects of BECN1 siRNA and ATG7 siRNA on the increase of colony formation induced by EEF2K silencing. HT-29 or HCT-116 cells were transfected with si BECN1 , si ATG7 , si EEF2K , si BECN1 plus si EEF2K , or si ATG7 plus si EEF2K . After 48 h transfection, cells were seeded into 6-well plates at the density of 150 cells per well, incubated at 37 °C for 12 to 14 d and then stained with crystal violet (0.5% w/v). Colonies with 50 or more cells were counted. ( C ) The effects of E64d and pepstatin A on cell viability in EEF2K silenced cells. HT-29 or HCT-116 cells were transfected with nontargeting control siRNA or EEF2K siRNA. After 3 h transfection, cells were treated with vehicle (0.1% DMSO), or 10 μg/ml E64d and pepstatin A for 45 h and then assessed by the MTT assay. All data shown are expressed as the means ± SEM of 3 independent experiments. * P < 0.05, $ P < 0.01, and # P < 0.001, vs. the EEF2K siRNA group (si EEF2K ; A–C ).

Article Snippet: A plasmid pDONR223-EEF2K containing full-length of human EEF2K coding region was obtained from Addgene (Addgene plasmid 23726, USA).

Techniques: Transfection, MTT Assay, Incubation, Staining, Control

Figure 5. Silencing of EEF2K does not potentiate the anticancer efficacy of MK-2206 against colon cancer cells. ( A ) Representative western blot analysis of phospho-EEF2 (Thr56; p-EEF2) and LC3-II expression in HT-29 cells treated with MK-2206 (0, 0.1, 0.5, 1, 5, 10 μM) for 24 h. The amounts of LC3-II and ACTB were quantified. ( B ) Representative western blot and densitometric analysis normalized to ACTB demonstrating the effect of EEF2K silencing on MK-2206 induced accumulation of LC3-II. HT-29 cells were transfected with nontargeting control siRNA (siCTL) or EEF2K siRNA (si EEF2K ) for 24 h and then treated with the vehicle (0.1% DMSO) or MK-2206 (5 μM) for 24 h. ( C ) HT-29 cells were transfected with control siRNA or EEF2K siRNA for 24 h and then treated with the vehicle (0.1% DMSO), 5 μM MK-2206, or 10 μM MK-2206 for 24 h. Cell viability was analyzed by the MTT assay. Results shown are expressed as the means ± SEM of 3 independent experiments. All quantitative data shown represent the means ± SEM of at least 3 independent experiments. # P < 0.001, vs. the control group (0 μM; A ) or the siCTL plus vehicle group ( B ).

Journal: Autophagy

Article Title: Silencing of EEF2K (eukaryotic elongation factor-2 kinase) reveals AMPK-ULK1-dependent autophagy in colon cancer cells

doi: 10.4161/auto.29164

Figure Lengend Snippet: Figure 5. Silencing of EEF2K does not potentiate the anticancer efficacy of MK-2206 against colon cancer cells. ( A ) Representative western blot analysis of phospho-EEF2 (Thr56; p-EEF2) and LC3-II expression in HT-29 cells treated with MK-2206 (0, 0.1, 0.5, 1, 5, 10 μM) for 24 h. The amounts of LC3-II and ACTB were quantified. ( B ) Representative western blot and densitometric analysis normalized to ACTB demonstrating the effect of EEF2K silencing on MK-2206 induced accumulation of LC3-II. HT-29 cells were transfected with nontargeting control siRNA (siCTL) or EEF2K siRNA (si EEF2K ) for 24 h and then treated with the vehicle (0.1% DMSO) or MK-2206 (5 μM) for 24 h. ( C ) HT-29 cells were transfected with control siRNA or EEF2K siRNA for 24 h and then treated with the vehicle (0.1% DMSO), 5 μM MK-2206, or 10 μM MK-2206 for 24 h. Cell viability was analyzed by the MTT assay. Results shown are expressed as the means ± SEM of 3 independent experiments. All quantitative data shown represent the means ± SEM of at least 3 independent experiments. # P < 0.001, vs. the control group (0 μM; A ) or the siCTL plus vehicle group ( B ).

Article Snippet: A plasmid pDONR223-EEF2K containing full-length of human EEF2K coding region was obtained from Addgene (Addgene plasmid 23726, USA).

Techniques: Western Blot, Expressing, Transfection, Control, MTT Assay

Figure 6. The AMPK-ULK1 pathway is required for autophagy induced by EEF2K silencing. ( A and B ) HT-29 or HCT-116 cells were transfected with nontargeting control siRNA (siCTL) or EEF2K siRNA (si EEF2K ) for 48 h. ( A ) Silencing of EEF2K reduces ATP level. After transfection, the ATP level was analyzed using the ATPlite Luminescence Assay Kit. ( B ) Silencing of EEF2K activates AMPK by phosphorylation at Thr172 and activates ULK1 by phosphorylation at Ser555 and dephosphorylation at Ser757, but does not inactivate MTOR. The protein levels of EEF2K, phospho-AMPKα (Thr172; p-AMPKα), AMPKα, phospho-ULK1 (Ser555; p-ULK1 (Ser555)), phospho-ULK1 (Ser757; p-ULK1 (Ser757)), ULK1, phospho-MTOR (Ser2448; p-MTOR), and ACTB were analyzed by western blot. ( C ) Representative western blot and densitometric analysis normalized to ACTB demonstrating the effect of cycloheximide on EEF2K silencing-induced LC3-II accumulation. HT-29 or HCT-116 cells were transfected with nontargeting control siRNA or EEF2K siRNA. At 3 h after transfection, cells were treated with 10 μg/ml cycloheximide (CHX) for 45 h. ( D–G ) HT-29 cells were transfected with control siRNA, EEF2K siRNA, PRKAA1 and PRKAA2/AMPKα siRNA (si AMPKα ), ULK1 siRNA (si ULK1 ), siEEF2K plus si AMPKα , or si EEF2K plus si ULK1 for 48 h. ( D ) Representative western blot demonstrating the effects of AMPKα siRNA on phospho-ULK1 (Ser555; p-ULK1) and LC3-II levels induced by EEF2K silencing. Densitometric analysis normalized to ACTB demonstrating the effect of AMPKα siRNA on LC3-II levels induced by EEF2K silencing. ( E ) Representative western blot and densitometric analysis normalized to ACTB demonstrating the effect of ULK1 siRNA on LC3-II levels induced by EEF2K silencing. ( F ) The effects of AMPKα siRNA and ULK1 siRNA on LC3 dots accumulation induced by EEF2K silencing. The average number of LC3 dots per cell was counted in more than 5 fields with at least 100 cells for each group and expressed as the means ± SEM of 3 independent experiments. # P < 0.001, vs. the EEF2K siRNA group (si EEF2K ). ( G ) The effects of AMPKα siRNA and ULK1 siRNA on the increase of cell viability induced by EEF2K silencing. Cell viability was analyzed by MTT assay. ( H and I ) The effect of EEF2K silencing on ROS generation. HT-29 cells were transfected with control siRNA or EEF2K siRNA for 48 h, and treated with 20 μM DCFDA for 30 min. ROS levels were detected by fluorescence microscopy ( H ) or by flow cytometry ( I ). H 2 O 2 (1 mM) treatment for 2 h was used as a positive control group. Scale bar: 20 μm. All quantitative data shown represent the means ± SEM of at least 3 independent experiments. * P < 0.05, $ P < 0.01 and # P < 0.001, vs. the siCTL group (for A ), or the EEF2K siRNA group (si EEF2K ; for C, D, E, and G ).

Journal: Autophagy

Article Title: Silencing of EEF2K (eukaryotic elongation factor-2 kinase) reveals AMPK-ULK1-dependent autophagy in colon cancer cells

doi: 10.4161/auto.29164

Figure Lengend Snippet: Figure 6. The AMPK-ULK1 pathway is required for autophagy induced by EEF2K silencing. ( A and B ) HT-29 or HCT-116 cells were transfected with nontargeting control siRNA (siCTL) or EEF2K siRNA (si EEF2K ) for 48 h. ( A ) Silencing of EEF2K reduces ATP level. After transfection, the ATP level was analyzed using the ATPlite Luminescence Assay Kit. ( B ) Silencing of EEF2K activates AMPK by phosphorylation at Thr172 and activates ULK1 by phosphorylation at Ser555 and dephosphorylation at Ser757, but does not inactivate MTOR. The protein levels of EEF2K, phospho-AMPKα (Thr172; p-AMPKα), AMPKα, phospho-ULK1 (Ser555; p-ULK1 (Ser555)), phospho-ULK1 (Ser757; p-ULK1 (Ser757)), ULK1, phospho-MTOR (Ser2448; p-MTOR), and ACTB were analyzed by western blot. ( C ) Representative western blot and densitometric analysis normalized to ACTB demonstrating the effect of cycloheximide on EEF2K silencing-induced LC3-II accumulation. HT-29 or HCT-116 cells were transfected with nontargeting control siRNA or EEF2K siRNA. At 3 h after transfection, cells were treated with 10 μg/ml cycloheximide (CHX) for 45 h. ( D–G ) HT-29 cells were transfected with control siRNA, EEF2K siRNA, PRKAA1 and PRKAA2/AMPKα siRNA (si AMPKα ), ULK1 siRNA (si ULK1 ), siEEF2K plus si AMPKα , or si EEF2K plus si ULK1 for 48 h. ( D ) Representative western blot demonstrating the effects of AMPKα siRNA on phospho-ULK1 (Ser555; p-ULK1) and LC3-II levels induced by EEF2K silencing. Densitometric analysis normalized to ACTB demonstrating the effect of AMPKα siRNA on LC3-II levels induced by EEF2K silencing. ( E ) Representative western blot and densitometric analysis normalized to ACTB demonstrating the effect of ULK1 siRNA on LC3-II levels induced by EEF2K silencing. ( F ) The effects of AMPKα siRNA and ULK1 siRNA on LC3 dots accumulation induced by EEF2K silencing. The average number of LC3 dots per cell was counted in more than 5 fields with at least 100 cells for each group and expressed as the means ± SEM of 3 independent experiments. # P < 0.001, vs. the EEF2K siRNA group (si EEF2K ). ( G ) The effects of AMPKα siRNA and ULK1 siRNA on the increase of cell viability induced by EEF2K silencing. Cell viability was analyzed by MTT assay. ( H and I ) The effect of EEF2K silencing on ROS generation. HT-29 cells were transfected with control siRNA or EEF2K siRNA for 48 h, and treated with 20 μM DCFDA for 30 min. ROS levels were detected by fluorescence microscopy ( H ) or by flow cytometry ( I ). H 2 O 2 (1 mM) treatment for 2 h was used as a positive control group. Scale bar: 20 μm. All quantitative data shown represent the means ± SEM of at least 3 independent experiments. * P < 0.05, $ P < 0.01 and # P < 0.001, vs. the siCTL group (for A ), or the EEF2K siRNA group (si EEF2K ; for C, D, E, and G ).

Article Snippet: A plasmid pDONR223-EEF2K containing full-length of human EEF2K coding region was obtained from Addgene (Addgene plasmid 23726, USA).

Techniques: Transfection, Control, Luminescence Assay, Phospho-proteomics, De-Phosphorylation Assay, Western Blot, MTT Assay, Fluorescence, Microscopy, Flow Cytometry, Positive Control

Figure 7. Signaling connections involved in autophagy pathways sensitive to EEF2K silencing in colon cancer cells. EEF2K can directly inactivate EEF2 by phosphorylation at Thr56, which negatively regulates peptide chain elongation. Silencing of EEF2K can upregulate the protein levels of BECN1 and ATG7. The upregulation of protein synthesis by EEF2K silencing can downregulate ATP level and increase AMP/ATP ratio. This in turn directly activates AMPKα by phosphorylation at Thr172 and then activates ULK1 by phosphorylation at Ser555 leading to autophagy. Autophagy induced by EEF2K silencing can promote human colon cancer cell proliferation. Inhibition of protein synthesis by cycloheximide can attenuate ULK1 activation and autophagy generation induced by EEF2K silencing. Arrows represent promotion events, blunt arrows indicate suppression events.

Journal: Autophagy

Article Title: Silencing of EEF2K (eukaryotic elongation factor-2 kinase) reveals AMPK-ULK1-dependent autophagy in colon cancer cells

doi: 10.4161/auto.29164

Figure Lengend Snippet: Figure 7. Signaling connections involved in autophagy pathways sensitive to EEF2K silencing in colon cancer cells. EEF2K can directly inactivate EEF2 by phosphorylation at Thr56, which negatively regulates peptide chain elongation. Silencing of EEF2K can upregulate the protein levels of BECN1 and ATG7. The upregulation of protein synthesis by EEF2K silencing can downregulate ATP level and increase AMP/ATP ratio. This in turn directly activates AMPKα by phosphorylation at Thr172 and then activates ULK1 by phosphorylation at Ser555 leading to autophagy. Autophagy induced by EEF2K silencing can promote human colon cancer cell proliferation. Inhibition of protein synthesis by cycloheximide can attenuate ULK1 activation and autophagy generation induced by EEF2K silencing. Arrows represent promotion events, blunt arrows indicate suppression events.

Article Snippet: A plasmid pDONR223-EEF2K containing full-length of human EEF2K coding region was obtained from Addgene (Addgene plasmid 23726, USA).

Techniques: Phospho-proteomics, Inhibition, Activation Assay

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