Journal: Cancer Biology & Therapy

Article Title: (−)-Guaiol regulates autophagic cell death depending on mTOR signaling in NSCLC

doi: 10.1080/15384047.2018.1451277

Figure Lengend Snippet: (−)-Guaiol induces autophagy by targeting mTOR pathways in NSCLC cells. A-B. Western blotting analysis of total protein from A549 (A) and H1299 (B) cells treated with or without (−)-Guaiol or (−)-Guaiol+MHY1485 in serum free medium for 24 h were conducted with indicated antibodies, taking GAPDH used as the internal control. The relative protein levels, which were used to statistical analysis, were calculated by normalizing the densitometry of treated cells to that of untreated cells.

Article Snippet: In the study, the following antibodies mTOR (MABS196), ULK1(MABC732), phospho-ULK1 Ser638 (p-ULK1 S638, MABC735) and ATG13 (ABC344), phospho-mTOR Ser2448 (p-mTOR S2448, 09–213), phospho-mTOR Ser2481(p-mTOR S2481, 09–343) were all purchased from Millipore.

Techniques: Western Blot, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: STAT3 suppresses the AMPKα / ULK1 ‐dependent induction of autophagy in glioblastoma cells

doi: 10.1111/jcmm.17421

Figure Lengend Snippet: STAT3 deletion activates autophagy through AMPKα‐ULK1‐TSC2 signalling pathways in MT330 cells. (A) Confluent EV MT330 cells, STAT3‐knockout cell line # 2 (KO2), STAT3‐knockout cell line # 3 (KO3), STAT3‐KO3 rescued with wild‐type (WT), and STAT3‐KO3 cells expressing Y705F‐STAT3 and S727A‐STAT3 mutants were exposed to Bafilomycin (Baf, 100 nM) for 3 h. Untreated (UT) cells served as controls. Total cell lysates were prepared and immunoblotted with indicated antibodies. (B) Quantification of the ratio of phospho‐STAT3,, and total‐STAT3 from three independent experiments. (C) Cell lysates were analysed for p‐AMPKα T172. Blots were stripped and probed for total‐AMPKα. (D) Quantification of the ratio of phosphorylated and total AMPKα shown in C. (E) Cell lysates were analysed for p‐ULK1 S555 and S638. Blots were stripped and probed for total‐ULK1. (F) Quantification of the ratio of phospho‐ULK1 S555 and total‐ULK1. (G) Western blotting of cell lysates with phospho‐TSC2 antibodies. Blots were stripped and probed with total‐TSC2 antibody. (H) Quantification of the ratio of phospho‐T1462‐ and total‐TSC2 and phospho‐S1387 and total‐TSC2

Article Snippet: Materials purchased include the following: Foetal bovine serum (Atlanta Biologicals); Enhanced chemiluminescence (ECL) Western blot detection system (Perkin Elmer, Inc.); Protease/Phosphatase Inhibitor Cocktail, cleaved active caspase‐3 (Asp 175); LC3‐I/LC3‐II; SQSTM1/p62, p‐Akt Ser473, HDAC‐6, phospho‐S6 Ribosomal protein Ser235/236, p‐STAT3 Y705, p‐STAT3 Ser727, Acetyl‐STAT3 Lys685, total‐STAT3, p‐AMPKα Thr172, Total‐ΑΜPΚα, p‐ULK1 Ser555, p‐ULK1 Ser638, Total‐ULK1, p‐TSC2 Ser1387, p‐TSC2 Ser1462, Total‐Tuberin/TSC2, Beclin‐1, BNIP3 and Cathepsin‐D antibodies (Cell Signalling Technology, Inc.); Alexa‐Fluor 488 conjugated, Alexa‐Fluor 647, and Cy3 conjugated secondary antibodies (Molecular Probes); Anti‐trimethyl STAT3 Lys180, and Bafilomycin A1, (EMD Biosciences/Millipore Corp.); ULK1 Inhibitor (MRT68921) (MedChemExpress).

Techniques: Knock-Out, Expressing, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: STAT3 suppresses the AMPKα / ULK1 ‐dependent induction of autophagy in glioblastoma cells

doi: 10.1111/jcmm.17421

Figure Lengend Snippet: STAT3 Represses autophagy in LN229 cells. (A) EV LN229 cells, STAT3‐knockout (KO), STAT3‐KO cells rescued with WT STAT3, and STAT3‐KO cells expressing Y705F‐STAT3 and S727A‐STAT3 phosphorylation‐defective mutants, were exposed to Bafilomycin (Baf, 100 nM) for 3 h or left untreated (UT). Total cell lysates were prepared and immunoblotted with indicated antibodies with β‐Actin serving as a loading control. (B) Densitometric analysis from n = 3 observations of the ratio of phosphorylated STAT3 to total‐STAT3 shown in A. (C) EV, STAT3‐KO and STAT3 mutant expressing lines with treated with or without 100 nM bafilomycin for 3 h. Cell lysates were immunoblotted for LC3‐I/II with Actin as a loading control. (D) Quantification of data shown in C. (E) Cell lysates were analysed for p‐AMPKα Thr172. Blots were stripped and probed for total‐AMPKα. (F) Quantification of the ratio of phosphorylated and total AMPKα shown in E. (G) Cell lysates were immunoblotted with the indicated antibodies. (H) Cell lysates were analysed for phospho‐ULK1 S555 and total‐ULK. (I) Quantification of data shown in H

Article Snippet: Materials purchased include the following: Foetal bovine serum (Atlanta Biologicals); Enhanced chemiluminescence (ECL) Western blot detection system (Perkin Elmer, Inc.); Protease/Phosphatase Inhibitor Cocktail, cleaved active caspase‐3 (Asp 175); LC3‐I/LC3‐II; SQSTM1/p62, p‐Akt Ser473, HDAC‐6, phospho‐S6 Ribosomal protein Ser235/236, p‐STAT3 Y705, p‐STAT3 Ser727, Acetyl‐STAT3 Lys685, total‐STAT3, p‐AMPKα Thr172, Total‐ΑΜPΚα, p‐ULK1 Ser555, p‐ULK1 Ser638, Total‐ULK1, p‐TSC2 Ser1387, p‐TSC2 Ser1462, Total‐Tuberin/TSC2, Beclin‐1, BNIP3 and Cathepsin‐D antibodies (Cell Signalling Technology, Inc.); Alexa‐Fluor 488 conjugated, Alexa‐Fluor 647, and Cy3 conjugated secondary antibodies (Molecular Probes); Anti‐trimethyl STAT3 Lys180, and Bafilomycin A1, (EMD Biosciences/Millipore Corp.); ULK1 Inhibitor (MRT68921) (MedChemExpress).

Techniques: Knock-Out, Expressing, Mutagenesis

Journal: Journal of Cellular and Molecular Medicine

Article Title: STAT3 suppresses the AMPKα / ULK1 ‐dependent induction of autophagy in glioblastoma cells

doi: 10.1111/jcmm.17421

Figure Lengend Snippet: STAT3‐Ko activates autophagy through mTOR‐independent but Prom1‐dependent signalling pathways in MT330 cells. (A) EV MT330 cells, STAT3‐KO, STAT3‐KO rescued with WT‐STAT3, and STAT3‐KO cells expressing Y705F (Y) and S727A (S) STAT3 mutants were treated with or without 100 nM bafilomycin for 3 h. Cell lysates were analysed for LC3‐I/II, p62 and CathepsinD. Quantification of (B) LC3‐II/Actin ratio and (C) p62/Actin ratio. (D) DMSO, Everolimus (RAD001) (10 μM) and RAD001 (10 μM) + Baf (100 nM) for 3 h. Cell lysates were immunoblotted with the indicated antibodies and beta actin was used as an internal loading control. (E) Quantification of phospho‐mTOR S2448 and total‐mTOR ratio. (F) Quantification of phospho‐S6 Ribosomal protein (Rbp) S235/236 and total‐S6 Rbp ratio. (G) Quantification of LC3‐II/Actin ratio presented in D. (H) EV, STAT3‐KO cells, KO3 rescued with WT‐STAT3, and KO3 cells expressing Y705F and S727A mutants were grown to confluence and treated with Baf (100 nM) for 3 h. Cell lysates were analysed by Western blotting using antibodies specific for STAT3, Prom1 and LC3‐I/II

Article Snippet: Materials purchased include the following: Foetal bovine serum (Atlanta Biologicals); Enhanced chemiluminescence (ECL) Western blot detection system (Perkin Elmer, Inc.); Protease/Phosphatase Inhibitor Cocktail, cleaved active caspase‐3 (Asp 175); LC3‐I/LC3‐II; SQSTM1/p62, p‐Akt Ser473, HDAC‐6, phospho‐S6 Ribosomal protein Ser235/236, p‐STAT3 Y705, p‐STAT3 Ser727, Acetyl‐STAT3 Lys685, total‐STAT3, p‐AMPKα Thr172, Total‐ΑΜPΚα, p‐ULK1 Ser555, p‐ULK1 Ser638, Total‐ULK1, p‐TSC2 Ser1387, p‐TSC2 Ser1462, Total‐Tuberin/TSC2, Beclin‐1, BNIP3 and Cathepsin‐D antibodies (Cell Signalling Technology, Inc.); Alexa‐Fluor 488 conjugated, Alexa‐Fluor 647, and Cy3 conjugated secondary antibodies (Molecular Probes); Anti‐trimethyl STAT3 Lys180, and Bafilomycin A1, (EMD Biosciences/Millipore Corp.); ULK1 Inhibitor (MRT68921) (MedChemExpress).

Techniques: Expressing, Western Blot