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

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

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

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

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

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

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

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