atg7 sirna  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc atg7 sirna
    Autophagy regulates TUBA acetylation. (A) Western blot analysis of protein acetylation in wild-type (WT), atg5−/-, and <t>atg7−/-</t> mouse embryonic fibroblasts (MEFs). n = 5, *P < 0.05 vs. WT. (B) WT MEFs were starved with Hank’s balanced salt solution (HBSS) for 3 h, and acetylated proteins were analyzed by western blotting. n = 5, *P < 0.05 vs. control (con). (C) Human aortic smooth muscle cells (HASMCs) were transfected with control <t>siRNA</t> (C-siRNA) or siRNA targeting ATG5 (ATG5-si) or ATG7 (ATG7-si) for 48 h. Protein acetylation was measured by western blotting. n = 5, *P< 0.05 vs. C-siRNA. (D) HASMCs were starved with HBSS for 3 h. Protein acetylation was analyzed by western blot. n = 5, *P< 0.05 vs. Con (E) Western blot analysis of acetylation of TUBA (Ac-TUBA) in WT, atg5−/-, and atg7−/- MEFs. n = 5, *P< 0.05 vs. WT. (F) Western blot analysis of Ac-TUBA in MEFs subjected to starvation. n = 5, *P < 0.05 vs. Con. (G) Levels of Ac-TUBA were determined by western blot in HASMCs transfected with C-siRNA, ATG5 siRNA, or ATG7 siRNA. n = 5, *P < 0.05 vs. C-siRNA. (H) Western blot analysis of Ac-TUBA in HASMCs subjected to starvation. n = 5, *P< 0.05 vs. Con. (I) Western blots detected the indicated proteins in HASMCs transfected with C-siRNA or BECN1 siRNA (BECN1-si). n = 5, *P< 0.05 vs. C-siRNA. (J) Western blot analysis of Ac-TUBA in HASMCs transfected with C-siRNA or ULK1 siRNA (ULK1-si). n = 5, *P< 0.05 vs. C-siRNA.
    Atg7 Sirna, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
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    1) Product Images from "Autophagic degradation of KAT2A/GCN5 promotes directional migration of vascular smooth muscle cells by reducing TUBA/α-tubulin acetylation"

    Article Title: Autophagic degradation of KAT2A/GCN5 promotes directional migration of vascular smooth muscle cells by reducing TUBA/α-tubulin acetylation

    Journal: Autophagy

    doi: 10.1080/15548627.2019.1707488

    Autophagy regulates TUBA acetylation. (A) Western blot analysis of protein acetylation in wild-type (WT), atg5−/-, and atg7−/- mouse embryonic fibroblasts (MEFs). n = 5, *P < 0.05 vs. WT. (B) WT MEFs were starved with Hank’s balanced salt solution (HBSS) for 3 h, and acetylated proteins were analyzed by western blotting. n = 5, *P < 0.05 vs. control (con). (C) Human aortic smooth muscle cells (HASMCs) were transfected with control siRNA (C-siRNA) or siRNA targeting ATG5 (ATG5-si) or ATG7 (ATG7-si) for 48 h. Protein acetylation was measured by western blotting. n = 5, *P< 0.05 vs. C-siRNA. (D) HASMCs were starved with HBSS for 3 h. Protein acetylation was analyzed by western blot. n = 5, *P< 0.05 vs. Con (E) Western blot analysis of acetylation of TUBA (Ac-TUBA) in WT, atg5−/-, and atg7−/- MEFs. n = 5, *P< 0.05 vs. WT. (F) Western blot analysis of Ac-TUBA in MEFs subjected to starvation. n = 5, *P < 0.05 vs. Con. (G) Levels of Ac-TUBA were determined by western blot in HASMCs transfected with C-siRNA, ATG5 siRNA, or ATG7 siRNA. n = 5, *P < 0.05 vs. C-siRNA. (H) Western blot analysis of Ac-TUBA in HASMCs subjected to starvation. n = 5, *P< 0.05 vs. Con. (I) Western blots detected the indicated proteins in HASMCs transfected with C-siRNA or BECN1 siRNA (BECN1-si). n = 5, *P< 0.05 vs. C-siRNA. (J) Western blot analysis of Ac-TUBA in HASMCs transfected with C-siRNA or ULK1 siRNA (ULK1-si). n = 5, *P< 0.05 vs. C-siRNA.
    Figure Legend Snippet: Autophagy regulates TUBA acetylation. (A) Western blot analysis of protein acetylation in wild-type (WT), atg5−/-, and atg7−/- mouse embryonic fibroblasts (MEFs). n = 5, *P < 0.05 vs. WT. (B) WT MEFs were starved with Hank’s balanced salt solution (HBSS) for 3 h, and acetylated proteins were analyzed by western blotting. n = 5, *P < 0.05 vs. control (con). (C) Human aortic smooth muscle cells (HASMCs) were transfected with control siRNA (C-siRNA) or siRNA targeting ATG5 (ATG5-si) or ATG7 (ATG7-si) for 48 h. Protein acetylation was measured by western blotting. n = 5, *P< 0.05 vs. C-siRNA. (D) HASMCs were starved with HBSS for 3 h. Protein acetylation was analyzed by western blot. n = 5, *P< 0.05 vs. Con (E) Western blot analysis of acetylation of TUBA (Ac-TUBA) in WT, atg5−/-, and atg7−/- MEFs. n = 5, *P< 0.05 vs. WT. (F) Western blot analysis of Ac-TUBA in MEFs subjected to starvation. n = 5, *P < 0.05 vs. Con. (G) Levels of Ac-TUBA were determined by western blot in HASMCs transfected with C-siRNA, ATG5 siRNA, or ATG7 siRNA. n = 5, *P < 0.05 vs. C-siRNA. (H) Western blot analysis of Ac-TUBA in HASMCs subjected to starvation. n = 5, *P< 0.05 vs. Con. (I) Western blots detected the indicated proteins in HASMCs transfected with C-siRNA or BECN1 siRNA (BECN1-si). n = 5, *P< 0.05 vs. C-siRNA. (J) Western blot analysis of Ac-TUBA in HASMCs transfected with C-siRNA or ULK1 siRNA (ULK1-si). n = 5, *P< 0.05 vs. C-siRNA.

    Techniques Used: Western Blot, Transfection

    KAT2A inhibits VSMC migration by acetylating TUBA. (A-C) HASMCs were transfected with control siRNA (C-siRNA) or KAT2A-siRNA (KAT2A-si) for 48 h. (A) KAT2A levels were analyzed by western blot. (B) Representative images of HASMC migration in the scratch wound assay. Scale bar: 1 mm. (C) Migration distances of HASMCs. n = 4, * P< 0.05 vs. C-siRNA. (D-F) HASMCs were transfected with Ad-GFP or Ad-KAT2A for 48 h. (D) Levels of KAT2A were analyzed by western blotting. (E) Cell migration was determined by transwell migration assays. Scale bar: 1 mm. (F) Migrated cells were quantified. n = 5, *P< 0.05 vs. C-siRNA. (G-I) HASMCs were transfected with control siRNA (C-siRNA), ATG7 siRNA, or ATG7 and KAT2A siRNA for 48 h. (G) Levels of acetylated TUBA (Ac-TUBA), KAT2A, and ATG7 in cell lysates were measured by western blotting. (H) Representative images of HASMC migration in the scratch wound assay. Scale bar: 1 mm. (I) Migration distances of HASMCs. n = 3, *P< 0.05 vs. C-siRNA. #P< 0.05 vs. ATG7-siRNA. (J) HASMCs were co-transfected with Ad-GFP/WT TUBA, Ad-KAT2A/WT TUBA, or Ad-KAT2A/TUBAK40R mutant for 48 h. Cell migration was determined by transwell migration assays. Scale bar: 1 mm. (K) Migrated cells were quantified. n = 5, ***P< 0.001 vs. Ad-GFP/WT TUBA; ###P< 0.001 vs. Ad-KAT2A/WT TUBA.
    Figure Legend Snippet: KAT2A inhibits VSMC migration by acetylating TUBA. (A-C) HASMCs were transfected with control siRNA (C-siRNA) or KAT2A-siRNA (KAT2A-si) for 48 h. (A) KAT2A levels were analyzed by western blot. (B) Representative images of HASMC migration in the scratch wound assay. Scale bar: 1 mm. (C) Migration distances of HASMCs. n = 4, * P< 0.05 vs. C-siRNA. (D-F) HASMCs were transfected with Ad-GFP or Ad-KAT2A for 48 h. (D) Levels of KAT2A were analyzed by western blotting. (E) Cell migration was determined by transwell migration assays. Scale bar: 1 mm. (F) Migrated cells were quantified. n = 5, *P< 0.05 vs. C-siRNA. (G-I) HASMCs were transfected with control siRNA (C-siRNA), ATG7 siRNA, or ATG7 and KAT2A siRNA for 48 h. (G) Levels of acetylated TUBA (Ac-TUBA), KAT2A, and ATG7 in cell lysates were measured by western blotting. (H) Representative images of HASMC migration in the scratch wound assay. Scale bar: 1 mm. (I) Migration distances of HASMCs. n = 3, *P< 0.05 vs. C-siRNA. #P< 0.05 vs. ATG7-siRNA. (J) HASMCs were co-transfected with Ad-GFP/WT TUBA, Ad-KAT2A/WT TUBA, or Ad-KAT2A/TUBAK40R mutant for 48 h. Cell migration was determined by transwell migration assays. Scale bar: 1 mm. (K) Migrated cells were quantified. n = 5, ***P< 0.001 vs. Ad-GFP/WT TUBA; ###P< 0.001 vs. Ad-KAT2A/WT TUBA.

    Techniques Used: Migration, Transfection, Western Blot, Scratch Wound Assay Assay, Mutagenesis

    Autophagy inhibition increases KAT2A protein levels. (A-C) HASMCs were transfected with control siRNA (C-siRNA), ATG5 siRNA (ATG5-si), or ATG7 siRNA (ATG7-si) for 48 h. Protein levels of KAT2A and LC3-I or LC3-II were analyzed by western blotting and densitometry. n = 3, *P< 0.05 vs. C-siRNA. (D) KAT2A mRNA was measured by quantitative real-time PCR. (E-G) Western blot and densitometry analysis of KAT2A and LC3-I or LC3-II protein levels in WT, atg5−/-, and atg7−/- MEFs. n = 3, **P< 0.01 vs. WT, ***P< 0.001 vs. WT. (H) Expression of KAT2A mRNA in WT, atg5−/-, and atg7−/- MEFs. (I-K) HASMCs were transfected with Ad-GFP, Ad-ATG5, or Ad-ATG7 for 48 h. Protein levels of KAT2A and LC3-I or LC3-II were evaluated by western blotting and densitometry. n = 3, *P< 0.05, **P< 0.01, ***P< 0.001 vs. ad-GFP. (L) WT and atg5−/- MEFs were starved with HBSS for the indicated time points. Protein levels of KAT2A, LC3-I or LC3-II, and ATG5 were analyzed by western blotting. (M) The LC3-II to LC3-I ratio was analyzed by densitometry. n = 3, **P< 0.01 vs. control. (N) KAT2A degradation was analyzed by densitometry. n = 3, *P< 0.05 vs. atg5−/- MEFs; #P< 0.05 vs. atg5−/- 0 h. (O) HeLa cells were transfected with plasmid encoding GFP or Flag-KAT2A for 48 h and then starved with HBSS for the indicated time points. Expression of Flag-KAT2A was determined by western blotting. (P) HASMCs were treated with chloroquine (CQ, 10 µM) or lactacystin (Lacta, 5 µM) for 24 h. Levels of KAT2A, SQSTM1, LC3-I or LC3-II and RUNX2 were analyzed by western blotting.
    Figure Legend Snippet: Autophagy inhibition increases KAT2A protein levels. (A-C) HASMCs were transfected with control siRNA (C-siRNA), ATG5 siRNA (ATG5-si), or ATG7 siRNA (ATG7-si) for 48 h. Protein levels of KAT2A and LC3-I or LC3-II were analyzed by western blotting and densitometry. n = 3, *P< 0.05 vs. C-siRNA. (D) KAT2A mRNA was measured by quantitative real-time PCR. (E-G) Western blot and densitometry analysis of KAT2A and LC3-I or LC3-II protein levels in WT, atg5−/-, and atg7−/- MEFs. n = 3, **P< 0.01 vs. WT, ***P< 0.001 vs. WT. (H) Expression of KAT2A mRNA in WT, atg5−/-, and atg7−/- MEFs. (I-K) HASMCs were transfected with Ad-GFP, Ad-ATG5, or Ad-ATG7 for 48 h. Protein levels of KAT2A and LC3-I or LC3-II were evaluated by western blotting and densitometry. n = 3, *P< 0.05, **P< 0.01, ***P< 0.001 vs. ad-GFP. (L) WT and atg5−/- MEFs were starved with HBSS for the indicated time points. Protein levels of KAT2A, LC3-I or LC3-II, and ATG5 were analyzed by western blotting. (M) The LC3-II to LC3-I ratio was analyzed by densitometry. n = 3, **P< 0.01 vs. control. (N) KAT2A degradation was analyzed by densitometry. n = 3, *P< 0.05 vs. atg5−/- MEFs; #P< 0.05 vs. atg5−/- 0 h. (O) HeLa cells were transfected with plasmid encoding GFP or Flag-KAT2A for 48 h and then starved with HBSS for the indicated time points. Expression of Flag-KAT2A was determined by western blotting. (P) HASMCs were treated with chloroquine (CQ, 10 µM) or lactacystin (Lacta, 5 µM) for 24 h. Levels of KAT2A, SQSTM1, LC3-I or LC3-II and RUNX2 were analyzed by western blotting.

    Techniques Used: Inhibition, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Plasmid Preparation

    Autophagy deficiency increases microtubule stability. (A) HASMCs were transfected with control siRNA (C-siRNA) or ATG7 siRNA (ATG7-si) for 48 h. TUBA and Ac-TUBA was stained by immunocytochemistry. Scale bar: 20 µm. (B) Quantitative analysis of TUBA and Ac-TUBA intensity. n = 3, ***P< 0.001. (C) HASMCs transfected with C-siRNA or ATG7-si were incubated at 0ºC for 0, 5, 10, 15, or 30 min. The morphology of microtubules was examined using immunostaining of TUBA and confocal microscope. Scale bar: 20 µm. (D) Quantitative analysis of microtubule intensity. n = 25/group, ***P< 0.001. (E) WT and atg5−/- MEFs were treated with nocodazole for 30 min, and then the drug was washed out to allow the microtubules to repolymerize for the indicated times. Acetylation of TUBA (Ac-TUBA) in cell lysates were analyzed by western blot. St, steady-state situation without nocodazole treatment. (F) Densitometry analysis of acetylation of TUBA levels. n = 3, *P< 0.05, **P< 0.01, ***P< 0.001.
    Figure Legend Snippet: Autophagy deficiency increases microtubule stability. (A) HASMCs were transfected with control siRNA (C-siRNA) or ATG7 siRNA (ATG7-si) for 48 h. TUBA and Ac-TUBA was stained by immunocytochemistry. Scale bar: 20 µm. (B) Quantitative analysis of TUBA and Ac-TUBA intensity. n = 3, ***P< 0.001. (C) HASMCs transfected with C-siRNA or ATG7-si were incubated at 0ºC for 0, 5, 10, 15, or 30 min. The morphology of microtubules was examined using immunostaining of TUBA and confocal microscope. Scale bar: 20 µm. (D) Quantitative analysis of microtubule intensity. n = 25/group, ***P< 0.001. (E) WT and atg5−/- MEFs were treated with nocodazole for 30 min, and then the drug was washed out to allow the microtubules to repolymerize for the indicated times. Acetylation of TUBA (Ac-TUBA) in cell lysates were analyzed by western blot. St, steady-state situation without nocodazole treatment. (F) Densitometry analysis of acetylation of TUBA levels. n = 3, *P< 0.05, **P< 0.01, ***P< 0.001.

    Techniques Used: Transfection, Staining, Immunocytochemistry, Incubation, Immunostaining, Microscopy, Western Blot

    Autophagy is required for polarization and directional migration of VMSCs in vitro. (A) Representative images of HASMCs stained with TUBG antibody in cells subjected to a scratch wound assay. The cells were treated with control siRNA or ATG7 siRNA for 48 h, and the images were captured 6 h after the scratch. Scale bar: 20 µm. The cell polarization was assessed by the angles (θ) between the lines of TUBG and the scratched line at the center of each nucleus as a marker for microtubule-organizing center (MTOC) reorientation. (B) Quantitation of the angles (θ). n = 100, *P< 0.01 vs. C-siRNA. (C) Images of HASMC stained with TUBA (green) and Ac-TUBA (red) antibodies in cells subjected to the scratch wound assay. Nuclei were stained with DAPI (blue). The cells were transfected with control siRNA or ATG7 siRNA. Scale bar: 50 µm. (D-G) HASMCs were transfected with control siRNA (C-siRNA), ATG7 siRNA (ATG7-si), or BECN1 siRNA (BECN1-si) for 48 h. (D) Representative images of HASMC migration in the scratch wound assay. Scale bar: 1 mm. (E) Migration distances of HASMCs. n = 4, **P< 0.01 vs. C-siRNA. (F) Cell migration was determined by transwell migration assays. Scale bar: 1 mm. (G) Migrated cells were quantified. n = 5, ***P< 0.001 vs. C-siRNA. (H-K) HASMCs were transfected with adenovirus encoding GFP, ATG5, or ATG7 for 48 h. (H) Representative images of HASMC migration in the scratch wound assay. Scale bar: 1 mm. (I) Migration distances of HASMCs. n = 4, *P< 0.05 vs. C-siRNA. (J) Cell migration was determined by transwell migration assays. Scale bar: 1 mm. (K) Migrated cells were quantified. n = 4, *P< 0.05 vs. C-siRNA.
    Figure Legend Snippet: Autophagy is required for polarization and directional migration of VMSCs in vitro. (A) Representative images of HASMCs stained with TUBG antibody in cells subjected to a scratch wound assay. The cells were treated with control siRNA or ATG7 siRNA for 48 h, and the images were captured 6 h after the scratch. Scale bar: 20 µm. The cell polarization was assessed by the angles (θ) between the lines of TUBG and the scratched line at the center of each nucleus as a marker for microtubule-organizing center (MTOC) reorientation. (B) Quantitation of the angles (θ). n = 100, *P< 0.01 vs. C-siRNA. (C) Images of HASMC stained with TUBA (green) and Ac-TUBA (red) antibodies in cells subjected to the scratch wound assay. Nuclei were stained with DAPI (blue). The cells were transfected with control siRNA or ATG7 siRNA. Scale bar: 50 µm. (D-G) HASMCs were transfected with control siRNA (C-siRNA), ATG7 siRNA (ATG7-si), or BECN1 siRNA (BECN1-si) for 48 h. (D) Representative images of HASMC migration in the scratch wound assay. Scale bar: 1 mm. (E) Migration distances of HASMCs. n = 4, **P< 0.01 vs. C-siRNA. (F) Cell migration was determined by transwell migration assays. Scale bar: 1 mm. (G) Migrated cells were quantified. n = 5, ***P< 0.001 vs. C-siRNA. (H-K) HASMCs were transfected with adenovirus encoding GFP, ATG5, or ATG7 for 48 h. (H) Representative images of HASMC migration in the scratch wound assay. Scale bar: 1 mm. (I) Migration distances of HASMCs. n = 4, *P< 0.05 vs. C-siRNA. (J) Cell migration was determined by transwell migration assays. Scale bar: 1 mm. (K) Migrated cells were quantified. n = 4, *P< 0.05 vs. C-siRNA.

    Techniques Used: Migration, In Vitro, Staining, Scratch Wound Assay Assay, Marker, Quantitation Assay, Transfection

    atg7 sirna  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Structured Review

    Cell Signaling Technology Inc atg7 sirna
    Autophagy regulates TUBA acetylation. (A) Western blot analysis of protein acetylation in wild-type (WT), atg5−/-, and <t>atg7−/-</t> mouse embryonic fibroblasts (MEFs). n = 5, *P < 0.05 vs. WT. (B) WT MEFs were starved with Hank’s balanced salt solution (HBSS) for 3 h, and acetylated proteins were analyzed by western blotting. n = 5, *P < 0.05 vs. control (con). (C) Human aortic smooth muscle cells (HASMCs) were transfected with control <t>siRNA</t> (C-siRNA) or siRNA targeting ATG5 (ATG5-si) or ATG7 (ATG7-si) for 48 h. Protein acetylation was measured by western blotting. n = 5, *P< 0.05 vs. C-siRNA. (D) HASMCs were starved with HBSS for 3 h. Protein acetylation was analyzed by western blot. n = 5, *P< 0.05 vs. Con (E) Western blot analysis of acetylation of TUBA (Ac-TUBA) in WT, atg5−/-, and atg7−/- MEFs. n = 5, *P< 0.05 vs. WT. (F) Western blot analysis of Ac-TUBA in MEFs subjected to starvation. n = 5, *P < 0.05 vs. Con. (G) Levels of Ac-TUBA were determined by western blot in HASMCs transfected with C-siRNA, ATG5 siRNA, or ATG7 siRNA. n = 5, *P < 0.05 vs. C-siRNA. (H) Western blot analysis of Ac-TUBA in HASMCs subjected to starvation. n = 5, *P< 0.05 vs. Con. (I) Western blots detected the indicated proteins in HASMCs transfected with C-siRNA or BECN1 siRNA (BECN1-si). n = 5, *P< 0.05 vs. C-siRNA. (J) Western blot analysis of Ac-TUBA in HASMCs transfected with C-siRNA or ULK1 siRNA (ULK1-si). n = 5, *P< 0.05 vs. C-siRNA.
    Atg7 Sirna, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/atg7 sirna/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    atg7 sirna - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Autophagic degradation of KAT2A/GCN5 promotes directional migration of vascular smooth muscle cells by reducing TUBA/α-tubulin acetylation"

    Article Title: Autophagic degradation of KAT2A/GCN5 promotes directional migration of vascular smooth muscle cells by reducing TUBA/α-tubulin acetylation

    Journal: Autophagy

    doi: 10.1080/15548627.2019.1707488

    Autophagy regulates TUBA acetylation. (A) Western blot analysis of protein acetylation in wild-type (WT), atg5−/-, and atg7−/- mouse embryonic fibroblasts (MEFs). n = 5, *P < 0.05 vs. WT. (B) WT MEFs were starved with Hank’s balanced salt solution (HBSS) for 3 h, and acetylated proteins were analyzed by western blotting. n = 5, *P < 0.05 vs. control (con). (C) Human aortic smooth muscle cells (HASMCs) were transfected with control siRNA (C-siRNA) or siRNA targeting ATG5 (ATG5-si) or ATG7 (ATG7-si) for 48 h. Protein acetylation was measured by western blotting. n = 5, *P< 0.05 vs. C-siRNA. (D) HASMCs were starved with HBSS for 3 h. Protein acetylation was analyzed by western blot. n = 5, *P< 0.05 vs. Con (E) Western blot analysis of acetylation of TUBA (Ac-TUBA) in WT, atg5−/-, and atg7−/- MEFs. n = 5, *P< 0.05 vs. WT. (F) Western blot analysis of Ac-TUBA in MEFs subjected to starvation. n = 5, *P < 0.05 vs. Con. (G) Levels of Ac-TUBA were determined by western blot in HASMCs transfected with C-siRNA, ATG5 siRNA, or ATG7 siRNA. n = 5, *P < 0.05 vs. C-siRNA. (H) Western blot analysis of Ac-TUBA in HASMCs subjected to starvation. n = 5, *P< 0.05 vs. Con. (I) Western blots detected the indicated proteins in HASMCs transfected with C-siRNA or BECN1 siRNA (BECN1-si). n = 5, *P< 0.05 vs. C-siRNA. (J) Western blot analysis of Ac-TUBA in HASMCs transfected with C-siRNA or ULK1 siRNA (ULK1-si). n = 5, *P< 0.05 vs. C-siRNA.
    Figure Legend Snippet: Autophagy regulates TUBA acetylation. (A) Western blot analysis of protein acetylation in wild-type (WT), atg5−/-, and atg7−/- mouse embryonic fibroblasts (MEFs). n = 5, *P < 0.05 vs. WT. (B) WT MEFs were starved with Hank’s balanced salt solution (HBSS) for 3 h, and acetylated proteins were analyzed by western blotting. n = 5, *P < 0.05 vs. control (con). (C) Human aortic smooth muscle cells (HASMCs) were transfected with control siRNA (C-siRNA) or siRNA targeting ATG5 (ATG5-si) or ATG7 (ATG7-si) for 48 h. Protein acetylation was measured by western blotting. n = 5, *P< 0.05 vs. C-siRNA. (D) HASMCs were starved with HBSS for 3 h. Protein acetylation was analyzed by western blot. n = 5, *P< 0.05 vs. Con (E) Western blot analysis of acetylation of TUBA (Ac-TUBA) in WT, atg5−/-, and atg7−/- MEFs. n = 5, *P< 0.05 vs. WT. (F) Western blot analysis of Ac-TUBA in MEFs subjected to starvation. n = 5, *P < 0.05 vs. Con. (G) Levels of Ac-TUBA were determined by western blot in HASMCs transfected with C-siRNA, ATG5 siRNA, or ATG7 siRNA. n = 5, *P < 0.05 vs. C-siRNA. (H) Western blot analysis of Ac-TUBA in HASMCs subjected to starvation. n = 5, *P< 0.05 vs. Con. (I) Western blots detected the indicated proteins in HASMCs transfected with C-siRNA or BECN1 siRNA (BECN1-si). n = 5, *P< 0.05 vs. C-siRNA. (J) Western blot analysis of Ac-TUBA in HASMCs transfected with C-siRNA or ULK1 siRNA (ULK1-si). n = 5, *P< 0.05 vs. C-siRNA.

    Techniques Used: Western Blot, Transfection

    KAT2A inhibits VSMC migration by acetylating TUBA. (A-C) HASMCs were transfected with control siRNA (C-siRNA) or KAT2A-siRNA (KAT2A-si) for 48 h. (A) KAT2A levels were analyzed by western blot. (B) Representative images of HASMC migration in the scratch wound assay. Scale bar: 1 mm. (C) Migration distances of HASMCs. n = 4, * P< 0.05 vs. C-siRNA. (D-F) HASMCs were transfected with Ad-GFP or Ad-KAT2A for 48 h. (D) Levels of KAT2A were analyzed by western blotting. (E) Cell migration was determined by transwell migration assays. Scale bar: 1 mm. (F) Migrated cells were quantified. n = 5, *P< 0.05 vs. C-siRNA. (G-I) HASMCs were transfected with control siRNA (C-siRNA), ATG7 siRNA, or ATG7 and KAT2A siRNA for 48 h. (G) Levels of acetylated TUBA (Ac-TUBA), KAT2A, and ATG7 in cell lysates were measured by western blotting. (H) Representative images of HASMC migration in the scratch wound assay. Scale bar: 1 mm. (I) Migration distances of HASMCs. n = 3, *P< 0.05 vs. C-siRNA. #P< 0.05 vs. ATG7-siRNA. (J) HASMCs were co-transfected with Ad-GFP/WT TUBA, Ad-KAT2A/WT TUBA, or Ad-KAT2A/TUBAK40R mutant for 48 h. Cell migration was determined by transwell migration assays. Scale bar: 1 mm. (K) Migrated cells were quantified. n = 5, ***P< 0.001 vs. Ad-GFP/WT TUBA; ###P< 0.001 vs. Ad-KAT2A/WT TUBA.
    Figure Legend Snippet: KAT2A inhibits VSMC migration by acetylating TUBA. (A-C) HASMCs were transfected with control siRNA (C-siRNA) or KAT2A-siRNA (KAT2A-si) for 48 h. (A) KAT2A levels were analyzed by western blot. (B) Representative images of HASMC migration in the scratch wound assay. Scale bar: 1 mm. (C) Migration distances of HASMCs. n = 4, * P< 0.05 vs. C-siRNA. (D-F) HASMCs were transfected with Ad-GFP or Ad-KAT2A for 48 h. (D) Levels of KAT2A were analyzed by western blotting. (E) Cell migration was determined by transwell migration assays. Scale bar: 1 mm. (F) Migrated cells were quantified. n = 5, *P< 0.05 vs. C-siRNA. (G-I) HASMCs were transfected with control siRNA (C-siRNA), ATG7 siRNA, or ATG7 and KAT2A siRNA for 48 h. (G) Levels of acetylated TUBA (Ac-TUBA), KAT2A, and ATG7 in cell lysates were measured by western blotting. (H) Representative images of HASMC migration in the scratch wound assay. Scale bar: 1 mm. (I) Migration distances of HASMCs. n = 3, *P< 0.05 vs. C-siRNA. #P< 0.05 vs. ATG7-siRNA. (J) HASMCs were co-transfected with Ad-GFP/WT TUBA, Ad-KAT2A/WT TUBA, or Ad-KAT2A/TUBAK40R mutant for 48 h. Cell migration was determined by transwell migration assays. Scale bar: 1 mm. (K) Migrated cells were quantified. n = 5, ***P< 0.001 vs. Ad-GFP/WT TUBA; ###P< 0.001 vs. Ad-KAT2A/WT TUBA.

    Techniques Used: Migration, Transfection, Western Blot, Scratch Wound Assay Assay, Mutagenesis

    Autophagy inhibition increases KAT2A protein levels. (A-C) HASMCs were transfected with control siRNA (C-siRNA), ATG5 siRNA (ATG5-si), or ATG7 siRNA (ATG7-si) for 48 h. Protein levels of KAT2A and LC3-I or LC3-II were analyzed by western blotting and densitometry. n = 3, *P< 0.05 vs. C-siRNA. (D) KAT2A mRNA was measured by quantitative real-time PCR. (E-G) Western blot and densitometry analysis of KAT2A and LC3-I or LC3-II protein levels in WT, atg5−/-, and atg7−/- MEFs. n = 3, **P< 0.01 vs. WT, ***P< 0.001 vs. WT. (H) Expression of KAT2A mRNA in WT, atg5−/-, and atg7−/- MEFs. (I-K) HASMCs were transfected with Ad-GFP, Ad-ATG5, or Ad-ATG7 for 48 h. Protein levels of KAT2A and LC3-I or LC3-II were evaluated by western blotting and densitometry. n = 3, *P< 0.05, **P< 0.01, ***P< 0.001 vs. ad-GFP. (L) WT and atg5−/- MEFs were starved with HBSS for the indicated time points. Protein levels of KAT2A, LC3-I or LC3-II, and ATG5 were analyzed by western blotting. (M) The LC3-II to LC3-I ratio was analyzed by densitometry. n = 3, **P< 0.01 vs. control. (N) KAT2A degradation was analyzed by densitometry. n = 3, *P< 0.05 vs. atg5−/- MEFs; #P< 0.05 vs. atg5−/- 0 h. (O) HeLa cells were transfected with plasmid encoding GFP or Flag-KAT2A for 48 h and then starved with HBSS for the indicated time points. Expression of Flag-KAT2A was determined by western blotting. (P) HASMCs were treated with chloroquine (CQ, 10 µM) or lactacystin (Lacta, 5 µM) for 24 h. Levels of KAT2A, SQSTM1, LC3-I or LC3-II and RUNX2 were analyzed by western blotting.
    Figure Legend Snippet: Autophagy inhibition increases KAT2A protein levels. (A-C) HASMCs were transfected with control siRNA (C-siRNA), ATG5 siRNA (ATG5-si), or ATG7 siRNA (ATG7-si) for 48 h. Protein levels of KAT2A and LC3-I or LC3-II were analyzed by western blotting and densitometry. n = 3, *P< 0.05 vs. C-siRNA. (D) KAT2A mRNA was measured by quantitative real-time PCR. (E-G) Western blot and densitometry analysis of KAT2A and LC3-I or LC3-II protein levels in WT, atg5−/-, and atg7−/- MEFs. n = 3, **P< 0.01 vs. WT, ***P< 0.001 vs. WT. (H) Expression of KAT2A mRNA in WT, atg5−/-, and atg7−/- MEFs. (I-K) HASMCs were transfected with Ad-GFP, Ad-ATG5, or Ad-ATG7 for 48 h. Protein levels of KAT2A and LC3-I or LC3-II were evaluated by western blotting and densitometry. n = 3, *P< 0.05, **P< 0.01, ***P< 0.001 vs. ad-GFP. (L) WT and atg5−/- MEFs were starved with HBSS for the indicated time points. Protein levels of KAT2A, LC3-I or LC3-II, and ATG5 were analyzed by western blotting. (M) The LC3-II to LC3-I ratio was analyzed by densitometry. n = 3, **P< 0.01 vs. control. (N) KAT2A degradation was analyzed by densitometry. n = 3, *P< 0.05 vs. atg5−/- MEFs; #P< 0.05 vs. atg5−/- 0 h. (O) HeLa cells were transfected with plasmid encoding GFP or Flag-KAT2A for 48 h and then starved with HBSS for the indicated time points. Expression of Flag-KAT2A was determined by western blotting. (P) HASMCs were treated with chloroquine (CQ, 10 µM) or lactacystin (Lacta, 5 µM) for 24 h. Levels of KAT2A, SQSTM1, LC3-I or LC3-II and RUNX2 were analyzed by western blotting.

    Techniques Used: Inhibition, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Plasmid Preparation

    Autophagy deficiency increases microtubule stability. (A) HASMCs were transfected with control siRNA (C-siRNA) or ATG7 siRNA (ATG7-si) for 48 h. TUBA and Ac-TUBA was stained by immunocytochemistry. Scale bar: 20 µm. (B) Quantitative analysis of TUBA and Ac-TUBA intensity. n = 3, ***P< 0.001. (C) HASMCs transfected with C-siRNA or ATG7-si were incubated at 0ºC for 0, 5, 10, 15, or 30 min. The morphology of microtubules was examined using immunostaining of TUBA and confocal microscope. Scale bar: 20 µm. (D) Quantitative analysis of microtubule intensity. n = 25/group, ***P< 0.001. (E) WT and atg5−/- MEFs were treated with nocodazole for 30 min, and then the drug was washed out to allow the microtubules to repolymerize for the indicated times. Acetylation of TUBA (Ac-TUBA) in cell lysates were analyzed by western blot. St, steady-state situation without nocodazole treatment. (F) Densitometry analysis of acetylation of TUBA levels. n = 3, *P< 0.05, **P< 0.01, ***P< 0.001.
    Figure Legend Snippet: Autophagy deficiency increases microtubule stability. (A) HASMCs were transfected with control siRNA (C-siRNA) or ATG7 siRNA (ATG7-si) for 48 h. TUBA and Ac-TUBA was stained by immunocytochemistry. Scale bar: 20 µm. (B) Quantitative analysis of TUBA and Ac-TUBA intensity. n = 3, ***P< 0.001. (C) HASMCs transfected with C-siRNA or ATG7-si were incubated at 0ºC for 0, 5, 10, 15, or 30 min. The morphology of microtubules was examined using immunostaining of TUBA and confocal microscope. Scale bar: 20 µm. (D) Quantitative analysis of microtubule intensity. n = 25/group, ***P< 0.001. (E) WT and atg5−/- MEFs were treated with nocodazole for 30 min, and then the drug was washed out to allow the microtubules to repolymerize for the indicated times. Acetylation of TUBA (Ac-TUBA) in cell lysates were analyzed by western blot. St, steady-state situation without nocodazole treatment. (F) Densitometry analysis of acetylation of TUBA levels. n = 3, *P< 0.05, **P< 0.01, ***P< 0.001.

    Techniques Used: Transfection, Staining, Immunocytochemistry, Incubation, Immunostaining, Microscopy, Western Blot

    Autophagy is required for polarization and directional migration of VMSCs in vitro. (A) Representative images of HASMCs stained with TUBG antibody in cells subjected to a scratch wound assay. The cells were treated with control siRNA or ATG7 siRNA for 48 h, and the images were captured 6 h after the scratch. Scale bar: 20 µm. The cell polarization was assessed by the angles (θ) between the lines of TUBG and the scratched line at the center of each nucleus as a marker for microtubule-organizing center (MTOC) reorientation. (B) Quantitation of the angles (θ). n = 100, *P< 0.01 vs. C-siRNA. (C) Images of HASMC stained with TUBA (green) and Ac-TUBA (red) antibodies in cells subjected to the scratch wound assay. Nuclei were stained with DAPI (blue). The cells were transfected with control siRNA or ATG7 siRNA. Scale bar: 50 µm. (D-G) HASMCs were transfected with control siRNA (C-siRNA), ATG7 siRNA (ATG7-si), or BECN1 siRNA (BECN1-si) for 48 h. (D) Representative images of HASMC migration in the scratch wound assay. Scale bar: 1 mm. (E) Migration distances of HASMCs. n = 4, **P< 0.01 vs. C-siRNA. (F) Cell migration was determined by transwell migration assays. Scale bar: 1 mm. (G) Migrated cells were quantified. n = 5, ***P< 0.001 vs. C-siRNA. (H-K) HASMCs were transfected with adenovirus encoding GFP, ATG5, or ATG7 for 48 h. (H) Representative images of HASMC migration in the scratch wound assay. Scale bar: 1 mm. (I) Migration distances of HASMCs. n = 4, *P< 0.05 vs. C-siRNA. (J) Cell migration was determined by transwell migration assays. Scale bar: 1 mm. (K) Migrated cells were quantified. n = 4, *P< 0.05 vs. C-siRNA.
    Figure Legend Snippet: Autophagy is required for polarization and directional migration of VMSCs in vitro. (A) Representative images of HASMCs stained with TUBG antibody in cells subjected to a scratch wound assay. The cells were treated with control siRNA or ATG7 siRNA for 48 h, and the images were captured 6 h after the scratch. Scale bar: 20 µm. The cell polarization was assessed by the angles (θ) between the lines of TUBG and the scratched line at the center of each nucleus as a marker for microtubule-organizing center (MTOC) reorientation. (B) Quantitation of the angles (θ). n = 100, *P< 0.01 vs. C-siRNA. (C) Images of HASMC stained with TUBA (green) and Ac-TUBA (red) antibodies in cells subjected to the scratch wound assay. Nuclei were stained with DAPI (blue). The cells were transfected with control siRNA or ATG7 siRNA. Scale bar: 50 µm. (D-G) HASMCs were transfected with control siRNA (C-siRNA), ATG7 siRNA (ATG7-si), or BECN1 siRNA (BECN1-si) for 48 h. (D) Representative images of HASMC migration in the scratch wound assay. Scale bar: 1 mm. (E) Migration distances of HASMCs. n = 4, **P< 0.01 vs. C-siRNA. (F) Cell migration was determined by transwell migration assays. Scale bar: 1 mm. (G) Migrated cells were quantified. n = 5, ***P< 0.001 vs. C-siRNA. (H-K) HASMCs were transfected with adenovirus encoding GFP, ATG5, or ATG7 for 48 h. (H) Representative images of HASMC migration in the scratch wound assay. Scale bar: 1 mm. (I) Migration distances of HASMCs. n = 4, *P< 0.05 vs. C-siRNA. (J) Cell migration was determined by transwell migration assays. Scale bar: 1 mm. (K) Migrated cells were quantified. n = 4, *P< 0.05 vs. C-siRNA.

    Techniques Used: Migration, In Vitro, Staining, Scratch Wound Assay Assay, Marker, Quantitation Assay, Transfection

    siatg7  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc siatg7
    a Significantly enriched pathways in lomitapide-treated HCT116 cells compared with vehicle-treated cells identified through KEEG analysis. b Volcano plot showing significant gene expression changes in response to lomitapide treatment in HCT116 cells. c HT29 cells were transfected with GFP-LC3 plasmid for 24 h, and treated with 5 μM lomitapide for another 24 h. GFP-LC3 puncta was visualized by a confocal microscope. Scale bar: 20 μm. d Cell viability was measured in HT29 cells treated with 5 μM lomitapide in the absence or presence of 100 nM bafilomycin for 24 h. e HT29 cells were treated with 5 μM lomitapide in the absence or presence of 1 mM 3-MA for 24 h. LC3 levels were measured by immunoblotting to assess autophagy induction. f si-control and <t>siATG7–transfected</t> HT29 cells were treated with 5 μM lomitapide for 24 h. LC3 levels were measured by immunoblotting to assess autophagy induction.
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    1) Product Images from "Lomitapide, a cholesterol-lowering drug, is an anticancer agent that induces autophagic cell death via inhibiting mTOR"

    Article Title: Lomitapide, a cholesterol-lowering drug, is an anticancer agent that induces autophagic cell death via inhibiting mTOR

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-022-05039-6

    a Significantly enriched pathways in lomitapide-treated HCT116 cells compared with vehicle-treated cells identified through KEEG analysis. b Volcano plot showing significant gene expression changes in response to lomitapide treatment in HCT116 cells. c HT29 cells were transfected with GFP-LC3 plasmid for 24 h, and treated with 5 μM lomitapide for another 24 h. GFP-LC3 puncta was visualized by a confocal microscope. Scale bar: 20 μm. d Cell viability was measured in HT29 cells treated with 5 μM lomitapide in the absence or presence of 100 nM bafilomycin for 24 h. e HT29 cells were treated with 5 μM lomitapide in the absence or presence of 1 mM 3-MA for 24 h. LC3 levels were measured by immunoblotting to assess autophagy induction. f si-control and siATG7–transfected HT29 cells were treated with 5 μM lomitapide for 24 h. LC3 levels were measured by immunoblotting to assess autophagy induction.
    Figure Legend Snippet: a Significantly enriched pathways in lomitapide-treated HCT116 cells compared with vehicle-treated cells identified through KEEG analysis. b Volcano plot showing significant gene expression changes in response to lomitapide treatment in HCT116 cells. c HT29 cells were transfected with GFP-LC3 plasmid for 24 h, and treated with 5 μM lomitapide for another 24 h. GFP-LC3 puncta was visualized by a confocal microscope. Scale bar: 20 μm. d Cell viability was measured in HT29 cells treated with 5 μM lomitapide in the absence or presence of 100 nM bafilomycin for 24 h. e HT29 cells were treated with 5 μM lomitapide in the absence or presence of 1 mM 3-MA for 24 h. LC3 levels were measured by immunoblotting to assess autophagy induction. f si-control and siATG7–transfected HT29 cells were treated with 5 μM lomitapide for 24 h. LC3 levels were measured by immunoblotting to assess autophagy induction.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Microscopy, Western Blot

    atg7 sirna  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc atg7 sirna
    Etravirine induces AGR2 degradation by autophagy. ( a – d ) Western blot analysis showed the effect of etravirine on AGR2 expression and LC3B expression. ( e , f ) Immunofluorescence analysis showed the expressions of AGR2 and LC3B in A2780-ADR cells after treatment with 5 µM etravirine for 48 h and 50 µM CQ for 24 h by using Lion Heart FX automated microscope (40×). Scale bar 100 µm. ( g , h ) A2780 and A2780-ADR cells were exposed with wortmannin (PI3K inhibitor) 1 µM for 24 h prior to etravirine 10 µM for 72 h. ( i , k ) A2780 and A2780-ADR cells were pretreated with 50 µM CQ for 24 h and then treated with 10 µM etravirine for 72 h. ( l ) Knockdown of <t>ATG7</t> by 100 nM <t>siRNA</t> for 72 h in A2780 cells and then treated with etravirine 10 uM for 72 h, AGR2 and LC3B expression were detected by Western blot. Data are shown as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.
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    1) Product Images from "Inhibitory Effect of Etravirine, a Non-Nucleoside Reverse Transcriptase Inhibitor, via Anterior Gradient Protein 2 Homolog Degradation against Ovarian Cancer Metastasis"

    Article Title: Inhibitory Effect of Etravirine, a Non-Nucleoside Reverse Transcriptase Inhibitor, via Anterior Gradient Protein 2 Homolog Degradation against Ovarian Cancer Metastasis

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms23020944

    Etravirine induces AGR2 degradation by autophagy. ( a – d ) Western blot analysis showed the effect of etravirine on AGR2 expression and LC3B expression. ( e , f ) Immunofluorescence analysis showed the expressions of AGR2 and LC3B in A2780-ADR cells after treatment with 5 µM etravirine for 48 h and 50 µM CQ for 24 h by using Lion Heart FX automated microscope (40×). Scale bar 100 µm. ( g , h ) A2780 and A2780-ADR cells were exposed with wortmannin (PI3K inhibitor) 1 µM for 24 h prior to etravirine 10 µM for 72 h. ( i , k ) A2780 and A2780-ADR cells were pretreated with 50 µM CQ for 24 h and then treated with 10 µM etravirine for 72 h. ( l ) Knockdown of ATG7 by 100 nM siRNA for 72 h in A2780 cells and then treated with etravirine 10 uM for 72 h, AGR2 and LC3B expression were detected by Western blot. Data are shown as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.
    Figure Legend Snippet: Etravirine induces AGR2 degradation by autophagy. ( a – d ) Western blot analysis showed the effect of etravirine on AGR2 expression and LC3B expression. ( e , f ) Immunofluorescence analysis showed the expressions of AGR2 and LC3B in A2780-ADR cells after treatment with 5 µM etravirine for 48 h and 50 µM CQ for 24 h by using Lion Heart FX automated microscope (40×). Scale bar 100 µm. ( g , h ) A2780 and A2780-ADR cells were exposed with wortmannin (PI3K inhibitor) 1 µM for 24 h prior to etravirine 10 µM for 72 h. ( i , k ) A2780 and A2780-ADR cells were pretreated with 50 µM CQ for 24 h and then treated with 10 µM etravirine for 72 h. ( l ) Knockdown of ATG7 by 100 nM siRNA for 72 h in A2780 cells and then treated with etravirine 10 uM for 72 h, AGR2 and LC3B expression were detected by Western blot. Data are shown as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

    Techniques Used: Western Blot, Expressing, Immunofluorescence, Microscopy

    atg7 sirna  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc atg7 sirna
    Atg7 Sirna, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    capsule 14 73 7 11  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc capsule 14 73 7 11
    Capsule 14 73 7 11, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    sirna against atg7  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc sirna against atg7
    Effect of PE5 on autophagy regulatory proteins and autophagic cell death. ( A , B ) H460 cells were treated with PE5 and detected for LC3, p62, ATG5, and <t>ATG7</t> proteins by western blotting. The blots were reprobed with GAPDH to confirm equal loading of the protein samples. ( C ) H460 cells were pre-treated with wortmannin (1 µM) (an autophagic inhibitor) and treated with PE5 for 24 h. Expression of LC3 was analyzed by immunofluorescence staining. ( D ) H460 cells were treated with PE5 in the presence of wortmannin (1 µM) or rapamycin (200 nM). Cell viability was analyzed by MTT assay. ( E , F ) Cells were transfected with <t>siATG7</t> and treated with 50 µM PE5 for 24 h. Expression levels of each ATG and LC3 were assessed by western blot analysis. The blots were reprobed with GAPDH to confirm equal loading of the protein samples. Cell viability was assessed using the MTT assay at 48 h. ( G ) H460 cells were pre-treated with wortmannin (1 µM) (an autophagic inhibitor) or Z-VAD-FMK (20 μM) (apoptosis inhibitor) and treated with PE5 for 24 h. Expression of PARP and cleaved PARP were analyzed by western blot analysis. The blots were reprobed with GAPDH to confirm equal loading of the protein samples. Data represent the mean ± SD (n = 3) and (* p < 0.05, ** p < 0.01, compared with the untreated control) ( # p < 0.05, compared with PE5-treated alone and significantly different from siATG-transfected cells).
    Sirna Against Atg7, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Targeting AKT/mTOR and Bcl-2 for Autophagic and Apoptosis Cell Death in Lung Cancer: Novel Activity of a Polyphenol Compound"

    Article Title: Targeting AKT/mTOR and Bcl-2 for Autophagic and Apoptosis Cell Death in Lung Cancer: Novel Activity of a Polyphenol Compound

    Journal: Antioxidants

    doi: 10.3390/antiox10040534

    Effect of PE5 on autophagy regulatory proteins and autophagic cell death. ( A , B ) H460 cells were treated with PE5 and detected for LC3, p62, ATG5, and ATG7 proteins by western blotting. The blots were reprobed with GAPDH to confirm equal loading of the protein samples. ( C ) H460 cells were pre-treated with wortmannin (1 µM) (an autophagic inhibitor) and treated with PE5 for 24 h. Expression of LC3 was analyzed by immunofluorescence staining. ( D ) H460 cells were treated with PE5 in the presence of wortmannin (1 µM) or rapamycin (200 nM). Cell viability was analyzed by MTT assay. ( E , F ) Cells were transfected with siATG7 and treated with 50 µM PE5 for 24 h. Expression levels of each ATG and LC3 were assessed by western blot analysis. The blots were reprobed with GAPDH to confirm equal loading of the protein samples. Cell viability was assessed using the MTT assay at 48 h. ( G ) H460 cells were pre-treated with wortmannin (1 µM) (an autophagic inhibitor) or Z-VAD-FMK (20 μM) (apoptosis inhibitor) and treated with PE5 for 24 h. Expression of PARP and cleaved PARP were analyzed by western blot analysis. The blots were reprobed with GAPDH to confirm equal loading of the protein samples. Data represent the mean ± SD (n = 3) and (* p < 0.05, ** p < 0.01, compared with the untreated control) ( # p < 0.05, compared with PE5-treated alone and significantly different from siATG-transfected cells).
    Figure Legend Snippet: Effect of PE5 on autophagy regulatory proteins and autophagic cell death. ( A , B ) H460 cells were treated with PE5 and detected for LC3, p62, ATG5, and ATG7 proteins by western blotting. The blots were reprobed with GAPDH to confirm equal loading of the protein samples. ( C ) H460 cells were pre-treated with wortmannin (1 µM) (an autophagic inhibitor) and treated with PE5 for 24 h. Expression of LC3 was analyzed by immunofluorescence staining. ( D ) H460 cells were treated with PE5 in the presence of wortmannin (1 µM) or rapamycin (200 nM). Cell viability was analyzed by MTT assay. ( E , F ) Cells were transfected with siATG7 and treated with 50 µM PE5 for 24 h. Expression levels of each ATG and LC3 were assessed by western blot analysis. The blots were reprobed with GAPDH to confirm equal loading of the protein samples. Cell viability was assessed using the MTT assay at 48 h. ( G ) H460 cells were pre-treated with wortmannin (1 µM) (an autophagic inhibitor) or Z-VAD-FMK (20 μM) (apoptosis inhibitor) and treated with PE5 for 24 h. Expression of PARP and cleaved PARP were analyzed by western blot analysis. The blots were reprobed with GAPDH to confirm equal loading of the protein samples. Data represent the mean ± SD (n = 3) and (* p < 0.05, ** p < 0.01, compared with the untreated control) ( # p < 0.05, compared with PE5-treated alone and significantly different from siATG-transfected cells).

    Techniques Used: Western Blot, Expressing, Immunofluorescence, Staining, MTT Assay, Transfection

    sirna against atg7  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc sirna against atg7
    Altered energy homeostasis in mice lacking autophagy in SF1 neurons. (A) Body weight and (B) average food intake of <t>Atg7</t> loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP (n 4–6 per group) male mice. (C) Food intake of refed of adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice after fasting (n = 5 for each group). (D) Body composition and (E) leptin sensitivity of adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n = 5–6 for each group). (F) Serum leptin levels in fed and 12 h-fasted adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP (n = 3–5 per group) male mice. (G–I) Oxygen (O2) consumption and (J–L) carbon dioxide (CO2) production in (G, J) fed and (H, K) 12 h-fasted Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n = 5 for each group). Values are shown as mean ± SEM. ∗ P ≤ 0.05, and P ≤ 0.01 versus each group.
    Sirna Against Atg7, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Defective autophagy in Sf1 neurons perturbs the metabolic response to fasting and causes mitochondrial dysfunction"

    Article Title: Defective autophagy in Sf1 neurons perturbs the metabolic response to fasting and causes mitochondrial dysfunction

    Journal: Molecular Metabolism

    doi: 10.1016/j.molmet.2021.101186

    Altered energy homeostasis in mice lacking autophagy in SF1 neurons. (A) Body weight and (B) average food intake of Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP (n 4–6 per group) male mice. (C) Food intake of refed of adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice after fasting (n = 5 for each group). (D) Body composition and (E) leptin sensitivity of adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n = 5–6 for each group). (F) Serum leptin levels in fed and 12 h-fasted adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP (n = 3–5 per group) male mice. (G–I) Oxygen (O2) consumption and (J–L) carbon dioxide (CO2) production in (G, J) fed and (H, K) 12 h-fasted Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n = 5 for each group). Values are shown as mean ± SEM. ∗ P ≤ 0.05, and P ≤ 0.01 versus each group.
    Figure Legend Snippet: Altered energy homeostasis in mice lacking autophagy in SF1 neurons. (A) Body weight and (B) average food intake of Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP (n 4–6 per group) male mice. (C) Food intake of refed of adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice after fasting (n = 5 for each group). (D) Body composition and (E) leptin sensitivity of adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n = 5–6 for each group). (F) Serum leptin levels in fed and 12 h-fasted adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP (n = 3–5 per group) male mice. (G–I) Oxygen (O2) consumption and (J–L) carbon dioxide (CO2) production in (G, J) fed and (H, K) 12 h-fasted Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n = 5 for each group). Values are shown as mean ± SEM. ∗ P ≤ 0.05, and P ≤ 0.01 versus each group.

    Techniques Used:

    Glucose metabolism in Sf1 -Cre; Atg7 loxP/loxP mice. (A) Serum glucose and (B) insulin levels and (C) VMH insulin receptor (InsR) mRNA expression in fed and 12h-fasted adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP (n = 4–7 per group) male mice. (D) Glucose and (E) insulin tolerance tests of adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP (n = 4–6 per group) male mice. Values are shown as mean ± SEM. ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, and ∗∗ P ≤ 0.001 versus each group.
    Figure Legend Snippet: Glucose metabolism in Sf1 -Cre; Atg7 loxP/loxP mice. (A) Serum glucose and (B) insulin levels and (C) VMH insulin receptor (InsR) mRNA expression in fed and 12h-fasted adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP (n = 4–7 per group) male mice. (D) Glucose and (E) insulin tolerance tests of adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP (n = 4–6 per group) male mice. Values are shown as mean ± SEM. ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, and ∗∗ P ≤ 0.001 versus each group.

    Techniques Used: Expressing

    Fasting-induced hypothalamic neuronal activation is impaired in Sf1 -Cre; Atg7 loxP/loxP mice. (A–C) Representative images and quantification of cFos-immunoreactive cells in the (A) ventromedial (VMH), (B) arcuate (ARH), and (C) dorsomedial (DMH) nuclei of the hypothalamus of fed and fasted adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n = 3–7 per group). (D) Vglut2 , (E) Pomc, and (F) Npy mRNA expression in the ARH of adult fed and fasted Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n = 3–4 per group). Values are shown as mean ± SEM. ∗ P ≤ 0.05 versus each group.
    Figure Legend Snippet: Fasting-induced hypothalamic neuronal activation is impaired in Sf1 -Cre; Atg7 loxP/loxP mice. (A–C) Representative images and quantification of cFos-immunoreactive cells in the (A) ventromedial (VMH), (B) arcuate (ARH), and (C) dorsomedial (DMH) nuclei of the hypothalamus of fed and fasted adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n = 3–7 per group). (D) Vglut2 , (E) Pomc, and (F) Npy mRNA expression in the ARH of adult fed and fasted Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n = 3–4 per group). Values are shown as mean ± SEM. ∗ P ≤ 0.05 versus each group.

    Techniques Used: Activation Assay, Expressing

    Loss of autophagy in SF1 neurons causes altered mitochondrial morphology and function. (A) Representative electron microscopy images of VMH neurons, percentage of autophagy-mitochondria contacts, and mitochondria aspect ratio in adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n = 4 per group). (B) Representative images and quantification of MitoTracker labeling in primary cultures of hypothalamic neurons 1 day after transfection with Atg7 siRNA or scrambled siRNA (n = 3–4 per group). (C) Mitochondria-related gene mRNA levels in the VMH of adult (10-week-old) fed or 12 h-fasted mice (n = 3–5 per group). (D) Measurement of mitochondrial respiration in the mediobasal hypothalamus of adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n = 4–8 per group). Values are shown as mean ± SEM. ∗ P ≤ 0.05 versus each group.
    Figure Legend Snippet: Loss of autophagy in SF1 neurons causes altered mitochondrial morphology and function. (A) Representative electron microscopy images of VMH neurons, percentage of autophagy-mitochondria contacts, and mitochondria aspect ratio in adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n = 4 per group). (B) Representative images and quantification of MitoTracker labeling in primary cultures of hypothalamic neurons 1 day after transfection with Atg7 siRNA or scrambled siRNA (n = 3–4 per group). (C) Mitochondria-related gene mRNA levels in the VMH of adult (10-week-old) fed or 12 h-fasted mice (n = 3–5 per group). (D) Measurement of mitochondrial respiration in the mediobasal hypothalamus of adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n = 4–8 per group). Values are shown as mean ± SEM. ∗ P ≤ 0.05 versus each group.

    Techniques Used: Electron Microscopy, Labeling, Transfection

    sirna against atg7  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc sirna against atg7
    Representative images and quantification of (A) ubiquitin- and (B) p62-immunoreactivity in the ventromedial nucleus of the hypothalamus (VMH) of adult <t>Atg7</t> loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n = 2-3 per group). ARH, arcuate nucleus of the hypothalamus; V3, third ventricle. Scale bar, 50 μm. Values are shown as mean ± SEM. * P ≤ 0.05 versus Atg7 loxP/loxP .
    Sirna Against Atg7, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Defective Autophagy in Sf1 Neurons Perturbs the Metabolic Response to Fasting and Causes Mitochondrial Dysfunction"

    Article Title: Defective Autophagy in Sf1 Neurons Perturbs the Metabolic Response to Fasting and Causes Mitochondrial Dysfunction

    Journal: bioRxiv

    doi: 10.1101/2020.11.02.348789

    Representative images and quantification of (A) ubiquitin- and (B) p62-immunoreactivity in the ventromedial nucleus of the hypothalamus (VMH) of adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n = 2-3 per group). ARH, arcuate nucleus of the hypothalamus; V3, third ventricle. Scale bar, 50 μm. Values are shown as mean ± SEM. * P ≤ 0.05 versus Atg7 loxP/loxP .
    Figure Legend Snippet: Representative images and quantification of (A) ubiquitin- and (B) p62-immunoreactivity in the ventromedial nucleus of the hypothalamus (VMH) of adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n = 2-3 per group). ARH, arcuate nucleus of the hypothalamus; V3, third ventricle. Scale bar, 50 μm. Values are shown as mean ± SEM. * P ≤ 0.05 versus Atg7 loxP/loxP .

    Techniques Used:

    (A) Body weight and (B) average food intake of Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP (n 4-6 per group) male mice. (C) Food intake of refed of adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice after fasting (n = 5 for each group). (D) Body composition and (E) leptin sensitivity of adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n = 5-6 for each group). (F) Serum leptin levels in fed and 12h-fasted adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP (n = 3-5 per group) male mice. (G-I) Oxygen (O2) consumption and (J-L) carbon dioxide (CO2) production in (G, J) fed and (H, K) 12h-fasted Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n = 5 for each group). Values are shown as mean ± SEM. * P ≤ 0.05, and P ≤ 0.01 versus each group.
    Figure Legend Snippet: (A) Body weight and (B) average food intake of Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP (n 4-6 per group) male mice. (C) Food intake of refed of adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice after fasting (n = 5 for each group). (D) Body composition and (E) leptin sensitivity of adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n = 5-6 for each group). (F) Serum leptin levels in fed and 12h-fasted adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP (n = 3-5 per group) male mice. (G-I) Oxygen (O2) consumption and (J-L) carbon dioxide (CO2) production in (G, J) fed and (H, K) 12h-fasted Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n = 5 for each group). Values are shown as mean ± SEM. * P ≤ 0.05, and P ≤ 0.01 versus each group.

    Techniques Used:

    (A-C) Respiratory quotient and (D-F) heat production in (A, D) fed and (B, E) 12h-fasted Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n = 4 for each group). Values are shown as mean ± SEM. * P ≤ 0.05 versus each group.
    Figure Legend Snippet: (A-C) Respiratory quotient and (D-F) heat production in (A, D) fed and (B, E) 12h-fasted Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n = 4 for each group). Values are shown as mean ± SEM. * P ≤ 0.05 versus each group.

    Techniques Used:

    (A) Serum glucose and (B) insulin levels, and (C) VMH insulin receptor (InsR) mRNA expression in fed and 12h-fasted adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP (n = 4-7 per group) male mice. (D) Glucose and (E) insulin tolerance tests and area under the curves of adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP (n = 4-6 per group) male mice. Values are shown as mean ± SEM. * P ≤ 0.05, ** P ≤ 0.01, and ** P ≤ 0.001 versus each group.
    Figure Legend Snippet: (A) Serum glucose and (B) insulin levels, and (C) VMH insulin receptor (InsR) mRNA expression in fed and 12h-fasted adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP (n = 4-7 per group) male mice. (D) Glucose and (E) insulin tolerance tests and area under the curves of adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP (n = 4-6 per group) male mice. Values are shown as mean ± SEM. * P ≤ 0.05, ** P ≤ 0.01, and ** P ≤ 0.001 versus each group.

    Techniques Used: Expressing

    (A-C) Representative images and quantification of cFos-immunoreactive cells in the (A) ventromedial (VMH), (B) arcuate (ARH), and (C) dorsomedial (DMH) nuclei of the hypothalamus of fed and fasted adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n = 3-7 per group). (D) Vglut2 , (E) Pomc, and (F) Npy mRNA expression in the ARH of adult fed and fasted Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n = 3-4 per group). Values are shown as mean ± SEM. * P ≤ 0.05 versus each group.
    Figure Legend Snippet: (A-C) Representative images and quantification of cFos-immunoreactive cells in the (A) ventromedial (VMH), (B) arcuate (ARH), and (C) dorsomedial (DMH) nuclei of the hypothalamus of fed and fasted adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n = 3-7 per group). (D) Vglut2 , (E) Pomc, and (F) Npy mRNA expression in the ARH of adult fed and fasted Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n = 3-4 per group). Values are shown as mean ± SEM. * P ≤ 0.05 versus each group.

    Techniques Used: Expressing

    (A) Representative electron microscopy images of VMH neurons, percentage of autophagy-mitochondria contacts, and mitochondria aspect ratio in adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n= 4 per group). (B) Representative images and quantification of MitoTracker labeling in primary cultures of hypothalamic neurons 1 day after transfection with Atg7 siRNA or scrambled siRNA (n = 3-4 per group). (C) Mitochondria-related gene mRNA levels in the VMH of adult (10-week-old) fed or 12h-fasted mice (n = 3-5 per group). (D) Measurement of mitochondrial respiration in the mediobasal hypothalamus of adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n = 4-8 per group). Values are shown as mean ± SEM. * P ≤ 0.05 versus each group.
    Figure Legend Snippet: (A) Representative electron microscopy images of VMH neurons, percentage of autophagy-mitochondria contacts, and mitochondria aspect ratio in adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n= 4 per group). (B) Representative images and quantification of MitoTracker labeling in primary cultures of hypothalamic neurons 1 day after transfection with Atg7 siRNA or scrambled siRNA (n = 3-4 per group). (C) Mitochondria-related gene mRNA levels in the VMH of adult (10-week-old) fed or 12h-fasted mice (n = 3-5 per group). (D) Measurement of mitochondrial respiration in the mediobasal hypothalamus of adult Atg7 loxP/loxP and Sf1 -Cre; Atg7 loxP/loxP male mice (n = 4-8 per group). Values are shown as mean ± SEM. * P ≤ 0.05 versus each group.

    Techniques Used: Electron Microscopy, Labeling, Transfection

    siatg7  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc siatg7
    Siatg7, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    signalsilence atg7 sirna i  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc signalsilence atg7 sirna i
    FPPS inhibition potentiates cell growth inhibition via impairment of autophagy. HUVECs were treated with IBAN (100 µM) with or without chloroquine (25 µM) for 24 hours. (a,b) Morphological changes of HUVECs following treatments were visualized using an inverted microscope (scale bar: 20 µm). (c) Cell proliferation was assessed using an MTS assay. The formazan dye produced by viable cells was quantified by measuring absorbance at 490 nm. (d) HUVECs were transiently transfected with an <t>Atg7-specific</t> siRNA. HUVECs were subsequently treated with IBAN (100 µM) for 24 hours, and cell proliferation was assessed using an MTS assay. *P< 0.05, **P< 0.01. (e) Western blotting was used to confirm Atg7 knockdown. Histograms were used to display the results of western blotting analysis. **P < 0.01 versus control group.
    Signalsilence Atg7 Sirna I, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Activation of autophagy during farnesyl pyrophosphate synthase inhibition is mediated through PI3K/AKT/mTOR signaling"

    Article Title: Activation of autophagy during farnesyl pyrophosphate synthase inhibition is mediated through PI3K/AKT/mTOR signaling

    Journal: The Journal of International Medical Research

    doi: 10.1177/0300060519875371

    FPPS inhibition potentiates cell growth inhibition via impairment of autophagy. HUVECs were treated with IBAN (100 µM) with or without chloroquine (25 µM) for 24 hours. (a,b) Morphological changes of HUVECs following treatments were visualized using an inverted microscope (scale bar: 20 µm). (c) Cell proliferation was assessed using an MTS assay. The formazan dye produced by viable cells was quantified by measuring absorbance at 490 nm. (d) HUVECs were transiently transfected with an Atg7-specific siRNA. HUVECs were subsequently treated with IBAN (100 µM) for 24 hours, and cell proliferation was assessed using an MTS assay. *P< 0.05, **P< 0.01. (e) Western blotting was used to confirm Atg7 knockdown. Histograms were used to display the results of western blotting analysis. **P < 0.01 versus control group.
    Figure Legend Snippet: FPPS inhibition potentiates cell growth inhibition via impairment of autophagy. HUVECs were treated with IBAN (100 µM) with or without chloroquine (25 µM) for 24 hours. (a,b) Morphological changes of HUVECs following treatments were visualized using an inverted microscope (scale bar: 20 µm). (c) Cell proliferation was assessed using an MTS assay. The formazan dye produced by viable cells was quantified by measuring absorbance at 490 nm. (d) HUVECs were transiently transfected with an Atg7-specific siRNA. HUVECs were subsequently treated with IBAN (100 µM) for 24 hours, and cell proliferation was assessed using an MTS assay. *P< 0.05, **P< 0.01. (e) Western blotting was used to confirm Atg7 knockdown. Histograms were used to display the results of western blotting analysis. **P < 0.01 versus control group.

    Techniques Used: Inhibition, Inverted Microscopy, MTS Assay, Produced, Transfection, Western Blot

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    Cell Signaling Technology Inc atg7 sirna
    Autophagy regulates TUBA acetylation. (A) Western blot analysis of protein acetylation in wild-type (WT), atg5−/-, and <t>atg7−/-</t> mouse embryonic fibroblasts (MEFs). n = 5, *P < 0.05 vs. WT. (B) WT MEFs were starved with Hank’s balanced salt solution (HBSS) for 3 h, and acetylated proteins were analyzed by western blotting. n = 5, *P < 0.05 vs. control (con). (C) Human aortic smooth muscle cells (HASMCs) were transfected with control <t>siRNA</t> (C-siRNA) or siRNA targeting ATG5 (ATG5-si) or ATG7 (ATG7-si) for 48 h. Protein acetylation was measured by western blotting. n = 5, *P< 0.05 vs. C-siRNA. (D) HASMCs were starved with HBSS for 3 h. Protein acetylation was analyzed by western blot. n = 5, *P< 0.05 vs. Con (E) Western blot analysis of acetylation of TUBA (Ac-TUBA) in WT, atg5−/-, and atg7−/- MEFs. n = 5, *P< 0.05 vs. WT. (F) Western blot analysis of Ac-TUBA in MEFs subjected to starvation. n = 5, *P < 0.05 vs. Con. (G) Levels of Ac-TUBA were determined by western blot in HASMCs transfected with C-siRNA, ATG5 siRNA, or ATG7 siRNA. n = 5, *P < 0.05 vs. C-siRNA. (H) Western blot analysis of Ac-TUBA in HASMCs subjected to starvation. n = 5, *P< 0.05 vs. Con. (I) Western blots detected the indicated proteins in HASMCs transfected with C-siRNA or BECN1 siRNA (BECN1-si). n = 5, *P< 0.05 vs. C-siRNA. (J) Western blot analysis of Ac-TUBA in HASMCs transfected with C-siRNA or ULK1 siRNA (ULK1-si). n = 5, *P< 0.05 vs. C-siRNA.
    Atg7 Sirna, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc siatg7
    a Significantly enriched pathways in lomitapide-treated HCT116 cells compared with vehicle-treated cells identified through KEEG analysis. b Volcano plot showing significant gene expression changes in response to lomitapide treatment in HCT116 cells. c HT29 cells were transfected with GFP-LC3 plasmid for 24 h, and treated with 5 μM lomitapide for another 24 h. GFP-LC3 puncta was visualized by a confocal microscope. Scale bar: 20 μm. d Cell viability was measured in HT29 cells treated with 5 μM lomitapide in the absence or presence of 100 nM bafilomycin for 24 h. e HT29 cells were treated with 5 μM lomitapide in the absence or presence of 1 mM 3-MA for 24 h. LC3 levels were measured by immunoblotting to assess autophagy induction. f si-control and <t>siATG7–transfected</t> HT29 cells were treated with 5 μM lomitapide for 24 h. LC3 levels were measured by immunoblotting to assess autophagy induction.
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    Cell Signaling Technology Inc capsule 14 73 7 11
    a Significantly enriched pathways in lomitapide-treated HCT116 cells compared with vehicle-treated cells identified through KEEG analysis. b Volcano plot showing significant gene expression changes in response to lomitapide treatment in HCT116 cells. c HT29 cells were transfected with GFP-LC3 plasmid for 24 h, and treated with 5 μM lomitapide for another 24 h. GFP-LC3 puncta was visualized by a confocal microscope. Scale bar: 20 μm. d Cell viability was measured in HT29 cells treated with 5 μM lomitapide in the absence or presence of 100 nM bafilomycin for 24 h. e HT29 cells were treated with 5 μM lomitapide in the absence or presence of 1 mM 3-MA for 24 h. LC3 levels were measured by immunoblotting to assess autophagy induction. f si-control and <t>siATG7–transfected</t> HT29 cells were treated with 5 μM lomitapide for 24 h. LC3 levels were measured by immunoblotting to assess autophagy induction.
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    Cell Signaling Technology Inc sirna against atg7
    Effect of PE5 on autophagy regulatory proteins and autophagic cell death. ( A , B ) H460 cells were treated with PE5 and detected for LC3, p62, ATG5, and <t>ATG7</t> proteins by western blotting. The blots were reprobed with GAPDH to confirm equal loading of the protein samples. ( C ) H460 cells were pre-treated with wortmannin (1 µM) (an autophagic inhibitor) and treated with PE5 for 24 h. Expression of LC3 was analyzed by immunofluorescence staining. ( D ) H460 cells were treated with PE5 in the presence of wortmannin (1 µM) or rapamycin (200 nM). Cell viability was analyzed by MTT assay. ( E , F ) Cells were transfected with <t>siATG7</t> and treated with 50 µM PE5 for 24 h. Expression levels of each ATG and LC3 were assessed by western blot analysis. The blots were reprobed with GAPDH to confirm equal loading of the protein samples. Cell viability was assessed using the MTT assay at 48 h. ( G ) H460 cells were pre-treated with wortmannin (1 µM) (an autophagic inhibitor) or Z-VAD-FMK (20 μM) (apoptosis inhibitor) and treated with PE5 for 24 h. Expression of PARP and cleaved PARP were analyzed by western blot analysis. The blots were reprobed with GAPDH to confirm equal loading of the protein samples. Data represent the mean ± SD (n = 3) and (* p < 0.05, ** p < 0.01, compared with the untreated control) ( # p < 0.05, compared with PE5-treated alone and significantly different from siATG-transfected cells).
    Sirna Against Atg7, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc signalsilence atg7 sirna i
    FPPS inhibition potentiates cell growth inhibition via impairment of autophagy. HUVECs were treated with IBAN (100 µM) with or without chloroquine (25 µM) for 24 hours. (a,b) Morphological changes of HUVECs following treatments were visualized using an inverted microscope (scale bar: 20 µm). (c) Cell proliferation was assessed using an MTS assay. The formazan dye produced by viable cells was quantified by measuring absorbance at 490 nm. (d) HUVECs were transiently transfected with an <t>Atg7-specific</t> siRNA. HUVECs were subsequently treated with IBAN (100 µM) for 24 hours, and cell proliferation was assessed using an MTS assay. *P< 0.05, **P< 0.01. (e) Western blotting was used to confirm Atg7 knockdown. Histograms were used to display the results of western blotting analysis. **P < 0.01 versus control group.
    Signalsilence Atg7 Sirna I, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Autophagy regulates TUBA acetylation. (A) Western blot analysis of protein acetylation in wild-type (WT), atg5−/-, and atg7−/- mouse embryonic fibroblasts (MEFs). n = 5, *P < 0.05 vs. WT. (B) WT MEFs were starved with Hank’s balanced salt solution (HBSS) for 3 h, and acetylated proteins were analyzed by western blotting. n = 5, *P < 0.05 vs. control (con). (C) Human aortic smooth muscle cells (HASMCs) were transfected with control siRNA (C-siRNA) or siRNA targeting ATG5 (ATG5-si) or ATG7 (ATG7-si) for 48 h. Protein acetylation was measured by western blotting. n = 5, *P< 0.05 vs. C-siRNA. (D) HASMCs were starved with HBSS for 3 h. Protein acetylation was analyzed by western blot. n = 5, *P< 0.05 vs. Con (E) Western blot analysis of acetylation of TUBA (Ac-TUBA) in WT, atg5−/-, and atg7−/- MEFs. n = 5, *P< 0.05 vs. WT. (F) Western blot analysis of Ac-TUBA in MEFs subjected to starvation. n = 5, *P < 0.05 vs. Con. (G) Levels of Ac-TUBA were determined by western blot in HASMCs transfected with C-siRNA, ATG5 siRNA, or ATG7 siRNA. n = 5, *P < 0.05 vs. C-siRNA. (H) Western blot analysis of Ac-TUBA in HASMCs subjected to starvation. n = 5, *P< 0.05 vs. Con. (I) Western blots detected the indicated proteins in HASMCs transfected with C-siRNA or BECN1 siRNA (BECN1-si). n = 5, *P< 0.05 vs. C-siRNA. (J) Western blot analysis of Ac-TUBA in HASMCs transfected with C-siRNA or ULK1 siRNA (ULK1-si). n = 5, *P< 0.05 vs. C-siRNA.

    Journal: Autophagy

    Article Title: Autophagic degradation of KAT2A/GCN5 promotes directional migration of vascular smooth muscle cells by reducing TUBA/α-tubulin acetylation

    doi: 10.1080/15548627.2019.1707488

    Figure Lengend Snippet: Autophagy regulates TUBA acetylation. (A) Western blot analysis of protein acetylation in wild-type (WT), atg5−/-, and atg7−/- mouse embryonic fibroblasts (MEFs). n = 5, *P < 0.05 vs. WT. (B) WT MEFs were starved with Hank’s balanced salt solution (HBSS) for 3 h, and acetylated proteins were analyzed by western blotting. n = 5, *P < 0.05 vs. control (con). (C) Human aortic smooth muscle cells (HASMCs) were transfected with control siRNA (C-siRNA) or siRNA targeting ATG5 (ATG5-si) or ATG7 (ATG7-si) for 48 h. Protein acetylation was measured by western blotting. n = 5, *P< 0.05 vs. C-siRNA. (D) HASMCs were starved with HBSS for 3 h. Protein acetylation was analyzed by western blot. n = 5, *P< 0.05 vs. Con (E) Western blot analysis of acetylation of TUBA (Ac-TUBA) in WT, atg5−/-, and atg7−/- MEFs. n = 5, *P< 0.05 vs. WT. (F) Western blot analysis of Ac-TUBA in MEFs subjected to starvation. n = 5, *P < 0.05 vs. Con. (G) Levels of Ac-TUBA were determined by western blot in HASMCs transfected with C-siRNA, ATG5 siRNA, or ATG7 siRNA. n = 5, *P < 0.05 vs. C-siRNA. (H) Western blot analysis of Ac-TUBA in HASMCs subjected to starvation. n = 5, *P< 0.05 vs. Con. (I) Western blots detected the indicated proteins in HASMCs transfected with C-siRNA or BECN1 siRNA (BECN1-si). n = 5, *P< 0.05 vs. C-siRNA. (J) Western blot analysis of Ac-TUBA in HASMCs transfected with C-siRNA or ULK1 siRNA (ULK1-si). n = 5, *P< 0.05 vs. C-siRNA.

    Article Snippet: KAT2A / GCN5 siRNA (Santa Cruz Biotechnology, 37946), ULK1 siRNA (Santa Cruz Biotechnology, 44849), ATG7 siRNA (Cell Signaling Technology, 6604), ATG5 siRNA (Cell Signaling Technology, 6345), SQSTM1 siRNA (Cell Signaling Technology, 6399).

    Techniques: Western Blot, Transfection

    KAT2A inhibits VSMC migration by acetylating TUBA. (A-C) HASMCs were transfected with control siRNA (C-siRNA) or KAT2A-siRNA (KAT2A-si) for 48 h. (A) KAT2A levels were analyzed by western blot. (B) Representative images of HASMC migration in the scratch wound assay. Scale bar: 1 mm. (C) Migration distances of HASMCs. n = 4, * P< 0.05 vs. C-siRNA. (D-F) HASMCs were transfected with Ad-GFP or Ad-KAT2A for 48 h. (D) Levels of KAT2A were analyzed by western blotting. (E) Cell migration was determined by transwell migration assays. Scale bar: 1 mm. (F) Migrated cells were quantified. n = 5, *P< 0.05 vs. C-siRNA. (G-I) HASMCs were transfected with control siRNA (C-siRNA), ATG7 siRNA, or ATG7 and KAT2A siRNA for 48 h. (G) Levels of acetylated TUBA (Ac-TUBA), KAT2A, and ATG7 in cell lysates were measured by western blotting. (H) Representative images of HASMC migration in the scratch wound assay. Scale bar: 1 mm. (I) Migration distances of HASMCs. n = 3, *P< 0.05 vs. C-siRNA. #P< 0.05 vs. ATG7-siRNA. (J) HASMCs were co-transfected with Ad-GFP/WT TUBA, Ad-KAT2A/WT TUBA, or Ad-KAT2A/TUBAK40R mutant for 48 h. Cell migration was determined by transwell migration assays. Scale bar: 1 mm. (K) Migrated cells were quantified. n = 5, ***P< 0.001 vs. Ad-GFP/WT TUBA; ###P< 0.001 vs. Ad-KAT2A/WT TUBA.

    Journal: Autophagy

    Article Title: Autophagic degradation of KAT2A/GCN5 promotes directional migration of vascular smooth muscle cells by reducing TUBA/α-tubulin acetylation

    doi: 10.1080/15548627.2019.1707488

    Figure Lengend Snippet: KAT2A inhibits VSMC migration by acetylating TUBA. (A-C) HASMCs were transfected with control siRNA (C-siRNA) or KAT2A-siRNA (KAT2A-si) for 48 h. (A) KAT2A levels were analyzed by western blot. (B) Representative images of HASMC migration in the scratch wound assay. Scale bar: 1 mm. (C) Migration distances of HASMCs. n = 4, * P< 0.05 vs. C-siRNA. (D-F) HASMCs were transfected with Ad-GFP or Ad-KAT2A for 48 h. (D) Levels of KAT2A were analyzed by western blotting. (E) Cell migration was determined by transwell migration assays. Scale bar: 1 mm. (F) Migrated cells were quantified. n = 5, *P< 0.05 vs. C-siRNA. (G-I) HASMCs were transfected with control siRNA (C-siRNA), ATG7 siRNA, or ATG7 and KAT2A siRNA for 48 h. (G) Levels of acetylated TUBA (Ac-TUBA), KAT2A, and ATG7 in cell lysates were measured by western blotting. (H) Representative images of HASMC migration in the scratch wound assay. Scale bar: 1 mm. (I) Migration distances of HASMCs. n = 3, *P< 0.05 vs. C-siRNA. #P< 0.05 vs. ATG7-siRNA. (J) HASMCs were co-transfected with Ad-GFP/WT TUBA, Ad-KAT2A/WT TUBA, or Ad-KAT2A/TUBAK40R mutant for 48 h. Cell migration was determined by transwell migration assays. Scale bar: 1 mm. (K) Migrated cells were quantified. n = 5, ***P< 0.001 vs. Ad-GFP/WT TUBA; ###P< 0.001 vs. Ad-KAT2A/WT TUBA.

    Article Snippet: KAT2A / GCN5 siRNA (Santa Cruz Biotechnology, 37946), ULK1 siRNA (Santa Cruz Biotechnology, 44849), ATG7 siRNA (Cell Signaling Technology, 6604), ATG5 siRNA (Cell Signaling Technology, 6345), SQSTM1 siRNA (Cell Signaling Technology, 6399).

    Techniques: Migration, Transfection, Western Blot, Scratch Wound Assay Assay, Mutagenesis

    Autophagy inhibition increases KAT2A protein levels. (A-C) HASMCs were transfected with control siRNA (C-siRNA), ATG5 siRNA (ATG5-si), or ATG7 siRNA (ATG7-si) for 48 h. Protein levels of KAT2A and LC3-I or LC3-II were analyzed by western blotting and densitometry. n = 3, *P< 0.05 vs. C-siRNA. (D) KAT2A mRNA was measured by quantitative real-time PCR. (E-G) Western blot and densitometry analysis of KAT2A and LC3-I or LC3-II protein levels in WT, atg5−/-, and atg7−/- MEFs. n = 3, **P< 0.01 vs. WT, ***P< 0.001 vs. WT. (H) Expression of KAT2A mRNA in WT, atg5−/-, and atg7−/- MEFs. (I-K) HASMCs were transfected with Ad-GFP, Ad-ATG5, or Ad-ATG7 for 48 h. Protein levels of KAT2A and LC3-I or LC3-II were evaluated by western blotting and densitometry. n = 3, *P< 0.05, **P< 0.01, ***P< 0.001 vs. ad-GFP. (L) WT and atg5−/- MEFs were starved with HBSS for the indicated time points. Protein levels of KAT2A, LC3-I or LC3-II, and ATG5 were analyzed by western blotting. (M) The LC3-II to LC3-I ratio was analyzed by densitometry. n = 3, **P< 0.01 vs. control. (N) KAT2A degradation was analyzed by densitometry. n = 3, *P< 0.05 vs. atg5−/- MEFs; #P< 0.05 vs. atg5−/- 0 h. (O) HeLa cells were transfected with plasmid encoding GFP or Flag-KAT2A for 48 h and then starved with HBSS for the indicated time points. Expression of Flag-KAT2A was determined by western blotting. (P) HASMCs were treated with chloroquine (CQ, 10 µM) or lactacystin (Lacta, 5 µM) for 24 h. Levels of KAT2A, SQSTM1, LC3-I or LC3-II and RUNX2 were analyzed by western blotting.

    Journal: Autophagy

    Article Title: Autophagic degradation of KAT2A/GCN5 promotes directional migration of vascular smooth muscle cells by reducing TUBA/α-tubulin acetylation

    doi: 10.1080/15548627.2019.1707488

    Figure Lengend Snippet: Autophagy inhibition increases KAT2A protein levels. (A-C) HASMCs were transfected with control siRNA (C-siRNA), ATG5 siRNA (ATG5-si), or ATG7 siRNA (ATG7-si) for 48 h. Protein levels of KAT2A and LC3-I or LC3-II were analyzed by western blotting and densitometry. n = 3, *P< 0.05 vs. C-siRNA. (D) KAT2A mRNA was measured by quantitative real-time PCR. (E-G) Western blot and densitometry analysis of KAT2A and LC3-I or LC3-II protein levels in WT, atg5−/-, and atg7−/- MEFs. n = 3, **P< 0.01 vs. WT, ***P< 0.001 vs. WT. (H) Expression of KAT2A mRNA in WT, atg5−/-, and atg7−/- MEFs. (I-K) HASMCs were transfected with Ad-GFP, Ad-ATG5, or Ad-ATG7 for 48 h. Protein levels of KAT2A and LC3-I or LC3-II were evaluated by western blotting and densitometry. n = 3, *P< 0.05, **P< 0.01, ***P< 0.001 vs. ad-GFP. (L) WT and atg5−/- MEFs were starved with HBSS for the indicated time points. Protein levels of KAT2A, LC3-I or LC3-II, and ATG5 were analyzed by western blotting. (M) The LC3-II to LC3-I ratio was analyzed by densitometry. n = 3, **P< 0.01 vs. control. (N) KAT2A degradation was analyzed by densitometry. n = 3, *P< 0.05 vs. atg5−/- MEFs; #P< 0.05 vs. atg5−/- 0 h. (O) HeLa cells were transfected with plasmid encoding GFP or Flag-KAT2A for 48 h and then starved with HBSS for the indicated time points. Expression of Flag-KAT2A was determined by western blotting. (P) HASMCs were treated with chloroquine (CQ, 10 µM) or lactacystin (Lacta, 5 µM) for 24 h. Levels of KAT2A, SQSTM1, LC3-I or LC3-II and RUNX2 were analyzed by western blotting.

    Article Snippet: KAT2A / GCN5 siRNA (Santa Cruz Biotechnology, 37946), ULK1 siRNA (Santa Cruz Biotechnology, 44849), ATG7 siRNA (Cell Signaling Technology, 6604), ATG5 siRNA (Cell Signaling Technology, 6345), SQSTM1 siRNA (Cell Signaling Technology, 6399).

    Techniques: Inhibition, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Plasmid Preparation

    Autophagy deficiency increases microtubule stability. (A) HASMCs were transfected with control siRNA (C-siRNA) or ATG7 siRNA (ATG7-si) for 48 h. TUBA and Ac-TUBA was stained by immunocytochemistry. Scale bar: 20 µm. (B) Quantitative analysis of TUBA and Ac-TUBA intensity. n = 3, ***P< 0.001. (C) HASMCs transfected with C-siRNA or ATG7-si were incubated at 0ºC for 0, 5, 10, 15, or 30 min. The morphology of microtubules was examined using immunostaining of TUBA and confocal microscope. Scale bar: 20 µm. (D) Quantitative analysis of microtubule intensity. n = 25/group, ***P< 0.001. (E) WT and atg5−/- MEFs were treated with nocodazole for 30 min, and then the drug was washed out to allow the microtubules to repolymerize for the indicated times. Acetylation of TUBA (Ac-TUBA) in cell lysates were analyzed by western blot. St, steady-state situation without nocodazole treatment. (F) Densitometry analysis of acetylation of TUBA levels. n = 3, *P< 0.05, **P< 0.01, ***P< 0.001.

    Journal: Autophagy

    Article Title: Autophagic degradation of KAT2A/GCN5 promotes directional migration of vascular smooth muscle cells by reducing TUBA/α-tubulin acetylation

    doi: 10.1080/15548627.2019.1707488

    Figure Lengend Snippet: Autophagy deficiency increases microtubule stability. (A) HASMCs were transfected with control siRNA (C-siRNA) or ATG7 siRNA (ATG7-si) for 48 h. TUBA and Ac-TUBA was stained by immunocytochemistry. Scale bar: 20 µm. (B) Quantitative analysis of TUBA and Ac-TUBA intensity. n = 3, ***P< 0.001. (C) HASMCs transfected with C-siRNA or ATG7-si were incubated at 0ºC for 0, 5, 10, 15, or 30 min. The morphology of microtubules was examined using immunostaining of TUBA and confocal microscope. Scale bar: 20 µm. (D) Quantitative analysis of microtubule intensity. n = 25/group, ***P< 0.001. (E) WT and atg5−/- MEFs were treated with nocodazole for 30 min, and then the drug was washed out to allow the microtubules to repolymerize for the indicated times. Acetylation of TUBA (Ac-TUBA) in cell lysates were analyzed by western blot. St, steady-state situation without nocodazole treatment. (F) Densitometry analysis of acetylation of TUBA levels. n = 3, *P< 0.05, **P< 0.01, ***P< 0.001.

    Article Snippet: KAT2A / GCN5 siRNA (Santa Cruz Biotechnology, 37946), ULK1 siRNA (Santa Cruz Biotechnology, 44849), ATG7 siRNA (Cell Signaling Technology, 6604), ATG5 siRNA (Cell Signaling Technology, 6345), SQSTM1 siRNA (Cell Signaling Technology, 6399).

    Techniques: Transfection, Staining, Immunocytochemistry, Incubation, Immunostaining, Microscopy, Western Blot

    Autophagy is required for polarization and directional migration of VMSCs in vitro. (A) Representative images of HASMCs stained with TUBG antibody in cells subjected to a scratch wound assay. The cells were treated with control siRNA or ATG7 siRNA for 48 h, and the images were captured 6 h after the scratch. Scale bar: 20 µm. The cell polarization was assessed by the angles (θ) between the lines of TUBG and the scratched line at the center of each nucleus as a marker for microtubule-organizing center (MTOC) reorientation. (B) Quantitation of the angles (θ). n = 100, *P< 0.01 vs. C-siRNA. (C) Images of HASMC stained with TUBA (green) and Ac-TUBA (red) antibodies in cells subjected to the scratch wound assay. Nuclei were stained with DAPI (blue). The cells were transfected with control siRNA or ATG7 siRNA. Scale bar: 50 µm. (D-G) HASMCs were transfected with control siRNA (C-siRNA), ATG7 siRNA (ATG7-si), or BECN1 siRNA (BECN1-si) for 48 h. (D) Representative images of HASMC migration in the scratch wound assay. Scale bar: 1 mm. (E) Migration distances of HASMCs. n = 4, **P< 0.01 vs. C-siRNA. (F) Cell migration was determined by transwell migration assays. Scale bar: 1 mm. (G) Migrated cells were quantified. n = 5, ***P< 0.001 vs. C-siRNA. (H-K) HASMCs were transfected with adenovirus encoding GFP, ATG5, or ATG7 for 48 h. (H) Representative images of HASMC migration in the scratch wound assay. Scale bar: 1 mm. (I) Migration distances of HASMCs. n = 4, *P< 0.05 vs. C-siRNA. (J) Cell migration was determined by transwell migration assays. Scale bar: 1 mm. (K) Migrated cells were quantified. n = 4, *P< 0.05 vs. C-siRNA.

    Journal: Autophagy

    Article Title: Autophagic degradation of KAT2A/GCN5 promotes directional migration of vascular smooth muscle cells by reducing TUBA/α-tubulin acetylation

    doi: 10.1080/15548627.2019.1707488

    Figure Lengend Snippet: Autophagy is required for polarization and directional migration of VMSCs in vitro. (A) Representative images of HASMCs stained with TUBG antibody in cells subjected to a scratch wound assay. The cells were treated with control siRNA or ATG7 siRNA for 48 h, and the images were captured 6 h after the scratch. Scale bar: 20 µm. The cell polarization was assessed by the angles (θ) between the lines of TUBG and the scratched line at the center of each nucleus as a marker for microtubule-organizing center (MTOC) reorientation. (B) Quantitation of the angles (θ). n = 100, *P< 0.01 vs. C-siRNA. (C) Images of HASMC stained with TUBA (green) and Ac-TUBA (red) antibodies in cells subjected to the scratch wound assay. Nuclei were stained with DAPI (blue). The cells were transfected with control siRNA or ATG7 siRNA. Scale bar: 50 µm. (D-G) HASMCs were transfected with control siRNA (C-siRNA), ATG7 siRNA (ATG7-si), or BECN1 siRNA (BECN1-si) for 48 h. (D) Representative images of HASMC migration in the scratch wound assay. Scale bar: 1 mm. (E) Migration distances of HASMCs. n = 4, **P< 0.01 vs. C-siRNA. (F) Cell migration was determined by transwell migration assays. Scale bar: 1 mm. (G) Migrated cells were quantified. n = 5, ***P< 0.001 vs. C-siRNA. (H-K) HASMCs were transfected with adenovirus encoding GFP, ATG5, or ATG7 for 48 h. (H) Representative images of HASMC migration in the scratch wound assay. Scale bar: 1 mm. (I) Migration distances of HASMCs. n = 4, *P< 0.05 vs. C-siRNA. (J) Cell migration was determined by transwell migration assays. Scale bar: 1 mm. (K) Migrated cells were quantified. n = 4, *P< 0.05 vs. C-siRNA.

    Article Snippet: KAT2A / GCN5 siRNA (Santa Cruz Biotechnology, 37946), ULK1 siRNA (Santa Cruz Biotechnology, 44849), ATG7 siRNA (Cell Signaling Technology, 6604), ATG5 siRNA (Cell Signaling Technology, 6345), SQSTM1 siRNA (Cell Signaling Technology, 6399).

    Techniques: Migration, In Vitro, Staining, Scratch Wound Assay Assay, Marker, Quantitation Assay, Transfection

    a Significantly enriched pathways in lomitapide-treated HCT116 cells compared with vehicle-treated cells identified through KEEG analysis. b Volcano plot showing significant gene expression changes in response to lomitapide treatment in HCT116 cells. c HT29 cells were transfected with GFP-LC3 plasmid for 24 h, and treated with 5 μM lomitapide for another 24 h. GFP-LC3 puncta was visualized by a confocal microscope. Scale bar: 20 μm. d Cell viability was measured in HT29 cells treated with 5 μM lomitapide in the absence or presence of 100 nM bafilomycin for 24 h. e HT29 cells were treated with 5 μM lomitapide in the absence or presence of 1 mM 3-MA for 24 h. LC3 levels were measured by immunoblotting to assess autophagy induction. f si-control and siATG7–transfected HT29 cells were treated with 5 μM lomitapide for 24 h. LC3 levels were measured by immunoblotting to assess autophagy induction.

    Journal: Cell Death & Disease

    Article Title: Lomitapide, a cholesterol-lowering drug, is an anticancer agent that induces autophagic cell death via inhibiting mTOR

    doi: 10.1038/s41419-022-05039-6

    Figure Lengend Snippet: a Significantly enriched pathways in lomitapide-treated HCT116 cells compared with vehicle-treated cells identified through KEEG analysis. b Volcano plot showing significant gene expression changes in response to lomitapide treatment in HCT116 cells. c HT29 cells were transfected with GFP-LC3 plasmid for 24 h, and treated with 5 μM lomitapide for another 24 h. GFP-LC3 puncta was visualized by a confocal microscope. Scale bar: 20 μm. d Cell viability was measured in HT29 cells treated with 5 μM lomitapide in the absence or presence of 100 nM bafilomycin for 24 h. e HT29 cells were treated with 5 μM lomitapide in the absence or presence of 1 mM 3-MA for 24 h. LC3 levels were measured by immunoblotting to assess autophagy induction. f si-control and siATG7–transfected HT29 cells were treated with 5 μM lomitapide for 24 h. LC3 levels were measured by immunoblotting to assess autophagy induction.

    Article Snippet: About 2 × 10 5 HT29 cells were inoculated in a six-well plate and incubated at 37 °C overnight, and then siRNAs were added for 48 h. For knock-down experiments, siATG7 (SignalSilence #6604; Cell Signaling Technology), siBeclin-1 (SignalSilence #6222; Cell Signaling Technology), siAMPK (siAMPK #1: 5′-CGACUAAGCCCAAAUCUUU-3′, siAMPK #2: 5′-ACCAUGAUUGAUGAUGAAGCCUUAA-3′) were used and then cells were treated with 0 or 5 μM concentration of lomitapide at 24 h prior to the endpoint.

    Techniques: Expressing, Transfection, Plasmid Preparation, Microscopy, Western Blot

    Effect of PE5 on autophagy regulatory proteins and autophagic cell death. ( A , B ) H460 cells were treated with PE5 and detected for LC3, p62, ATG5, and ATG7 proteins by western blotting. The blots were reprobed with GAPDH to confirm equal loading of the protein samples. ( C ) H460 cells were pre-treated with wortmannin (1 µM) (an autophagic inhibitor) and treated with PE5 for 24 h. Expression of LC3 was analyzed by immunofluorescence staining. ( D ) H460 cells were treated with PE5 in the presence of wortmannin (1 µM) or rapamycin (200 nM). Cell viability was analyzed by MTT assay. ( E , F ) Cells were transfected with siATG7 and treated with 50 µM PE5 for 24 h. Expression levels of each ATG and LC3 were assessed by western blot analysis. The blots were reprobed with GAPDH to confirm equal loading of the protein samples. Cell viability was assessed using the MTT assay at 48 h. ( G ) H460 cells were pre-treated with wortmannin (1 µM) (an autophagic inhibitor) or Z-VAD-FMK (20 μM) (apoptosis inhibitor) and treated with PE5 for 24 h. Expression of PARP and cleaved PARP were analyzed by western blot analysis. The blots were reprobed with GAPDH to confirm equal loading of the protein samples. Data represent the mean ± SD (n = 3) and (* p < 0.05, ** p < 0.01, compared with the untreated control) ( # p < 0.05, compared with PE5-treated alone and significantly different from siATG-transfected cells).

    Journal: Antioxidants

    Article Title: Targeting AKT/mTOR and Bcl-2 for Autophagic and Apoptosis Cell Death in Lung Cancer: Novel Activity of a Polyphenol Compound

    doi: 10.3390/antiox10040534

    Figure Lengend Snippet: Effect of PE5 on autophagy regulatory proteins and autophagic cell death. ( A , B ) H460 cells were treated with PE5 and detected for LC3, p62, ATG5, and ATG7 proteins by western blotting. The blots were reprobed with GAPDH to confirm equal loading of the protein samples. ( C ) H460 cells were pre-treated with wortmannin (1 µM) (an autophagic inhibitor) and treated with PE5 for 24 h. Expression of LC3 was analyzed by immunofluorescence staining. ( D ) H460 cells were treated with PE5 in the presence of wortmannin (1 µM) or rapamycin (200 nM). Cell viability was analyzed by MTT assay. ( E , F ) Cells were transfected with siATG7 and treated with 50 µM PE5 for 24 h. Expression levels of each ATG and LC3 were assessed by western blot analysis. The blots were reprobed with GAPDH to confirm equal loading of the protein samples. Cell viability was assessed using the MTT assay at 48 h. ( G ) H460 cells were pre-treated with wortmannin (1 µM) (an autophagic inhibitor) or Z-VAD-FMK (20 μM) (apoptosis inhibitor) and treated with PE5 for 24 h. Expression of PARP and cleaved PARP were analyzed by western blot analysis. The blots were reprobed with GAPDH to confirm equal loading of the protein samples. Data represent the mean ± SD (n = 3) and (* p < 0.05, ** p < 0.01, compared with the untreated control) ( # p < 0.05, compared with PE5-treated alone and significantly different from siATG-transfected cells).

    Article Snippet: The siRNA including siRNA control and siRNA against ATG7 were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA).

    Techniques: Western Blot, Expressing, Immunofluorescence, Staining, MTT Assay, Transfection

    FPPS inhibition potentiates cell growth inhibition via impairment of autophagy. HUVECs were treated with IBAN (100 µM) with or without chloroquine (25 µM) for 24 hours. (a,b) Morphological changes of HUVECs following treatments were visualized using an inverted microscope (scale bar: 20 µm). (c) Cell proliferation was assessed using an MTS assay. The formazan dye produced by viable cells was quantified by measuring absorbance at 490 nm. (d) HUVECs were transiently transfected with an Atg7-specific siRNA. HUVECs were subsequently treated with IBAN (100 µM) for 24 hours, and cell proliferation was assessed using an MTS assay. *P< 0.05, **P< 0.01. (e) Western blotting was used to confirm Atg7 knockdown. Histograms were used to display the results of western blotting analysis. **P < 0.01 versus control group.

    Journal: The Journal of International Medical Research

    Article Title: Activation of autophagy during farnesyl pyrophosphate synthase inhibition is mediated through PI3K/AKT/mTOR signaling

    doi: 10.1177/0300060519875371

    Figure Lengend Snippet: FPPS inhibition potentiates cell growth inhibition via impairment of autophagy. HUVECs were treated with IBAN (100 µM) with or without chloroquine (25 µM) for 24 hours. (a,b) Morphological changes of HUVECs following treatments were visualized using an inverted microscope (scale bar: 20 µm). (c) Cell proliferation was assessed using an MTS assay. The formazan dye produced by viable cells was quantified by measuring absorbance at 490 nm. (d) HUVECs were transiently transfected with an Atg7-specific siRNA. HUVECs were subsequently treated with IBAN (100 µM) for 24 hours, and cell proliferation was assessed using an MTS assay. *P< 0.05, **P< 0.01. (e) Western blotting was used to confirm Atg7 knockdown. Histograms were used to display the results of western blotting analysis. **P < 0.01 versus control group.

    Article Snippet: The siRNA for Atg7 was obtained from Cell Signaling Technology (SignalSilence® Atg7 siRNA I #6604).

    Techniques: Inhibition, Inverted Microscopy, MTS Assay, Produced, Transfection, Western Blot