Journal: Autophagy

Article Title: Piperlongumine restores the balance of autophagy and apoptosis by increasing BCL2 phosphorylation in rotenone-induced Parkinson disease models

doi: 10.1080/15548627.2017.1390636

Figure Lengend Snippet: PLG induces autophagy by increasing PtdIns3K complex activity. SK-N-SH cells and rat primary neurons were treated with rotenone. PLG was added 2 h later at 0.1 μM for 24 h. Rapamycin (Rap; 40 nM for 6 h), an autophagy agonist, served as a positive control. ( A, B ) Autophagy evaluated by counting fluorescent LC3 puncta in SK-N-SH cells ( A ) and primary neurons ( B ). ( C )Immunofluorescence analysis of SQSTM1 to evaluate autophagy in primary neurons. Nuclei were counterstained with DAPI. ( D ) Transfection of GFP-ZFYVE1 plasmid and immunofluorescence detection of WIPI2 to evaluate PtdIns3K complex activity in primary neurons. Nuclei were counterstained with DAPI. ( E, F ) Quantification of LC3 puncta in SK-N-SH cells ( E ) and primary neurons ( F ). ( G ) Quantification of SQSTM1 puncta in primary neurons. ( H, I ) Quantification of WIPI2 ( H ) and ZFYVE1 ( I ) puncte in primary neurons. Bar: 25 μm ( A, B ) and 10μm ( C, D ). Data are expressed as the mean ± SD (one-way analysis of variance). ###P<0.001 vs. control (Con), ***P<0.001 vs. rotenone (Rot) (n = 3).

Article Snippet: Antibodies against the following proteins were used in the study: ACTB (1:5000; Sigma-Aldrich, A5060), BAX (1:1000; Cell Signaling Technology, 2772), BCL2 (1:1000; Cell Signaling Technology, 3498), phosphorylated (p-)BCL2 (Ser70) (1:1000; Cell Signaling Technology, 2827), BECN1 (1:1000; Cell Signaling Technology, 3495), IgG (1:1000; Sigma-Aldrich, I5006), p-MAPK8/JNK1 (1:1000; Cell Signaling Technology, 4668 [we note that this antibody may react with phosphorylated MAPK1/3 and MAPK/p38]), MAPK8/JNK1 (1:1000; Cell Signaling Technology, 3708 [we note that this antibody may cross-react with MAPK9/JNK2]), LC3B (1:1,000; Novus Biologicals, NB100-2220), NFE2L2/NRF2 (1:1000; Cell Signaling Technology, 12721), SQSTM1 (1:1000; Cell Signaling Technology, 5114), TH (1:1000; Sigma-Aldrich, T2928), and WIPI2 (1:1000; Cell Signaling Technology, 8567).

Techniques: Activity Assay, Positive Control, Immunofluorescence, Transfection, Plasmid Preparation, Control

Journal: International Journal of Molecular Sciences

Article Title: ATG101 Degradation by HUWE1-Mediated Ubiquitination Impairs Autophagy and Reduces Survival in Cancer Cells

doi: 10.3390/ijms22179182

Figure Lengend Snippet: HUWE1 suppresses autophagy by regulating ATG101 and WIPI2 levels ( a ) HUWE1 KD enhanced GFP-LC3 puncta formation in MIA PaCa-2 cells. shCTL or shHUWE1 MIA PaCa-2 cells stably expressing GFP-LC3 were plated overnight, and subsequently incubated with rapamycin (0.5 μM) for 4 h prior to imaging. Cell imaging, quantification and statistical analysis were as described in d. * p < 0.05. ( b ) HUWE1 KD showed enhancement of autophagy activity as indicated by western blotting for autophagy marker proteins. Cell lysates from (a) were analyzed by western blot using the indicated antibodies for autophagy marker proteins. Quantification and statistical analysis were as in c. ( c ) ATG101 KD reversed the increase in GFP-LC3 puncta formation induced by shHUWE1 cells. shCTL or shHUWE1 MIA PaCa-2 cells stably expressing GFP-LC3 were transfected with ATG101 siRNA for 48 h then incubated with rapamycin (0.5 μM) for 4 h. Cell imaging, quantification and statistical analysis were as in d. Scale bar: 20 μm. Error bars indicate the mean ± SEM of three independent experiments. ** p < 0.01; *** p < 0.001. ( d ) Both ATG101 and WIPI2 double KD reversed the increase in autophagy flux induced by shHUWE1. shHUWE1 HeLa cells stably expressing mCherry-GFP-LC3 were transfected WIPI2 and/or ATG101 siRNA for 48 h, followed by incubation in amino acid-deprived medium (–AA) for 4 h. Cells were imaged under starvation conditions as in (c). Scale bar: 20 μm. mCherry puncta area (%) was normalized to Hoechst-stained area and is presented as a percentage on the quantification graph. Error bars indicate the mean ± SEM of three independent experiments. ** p < 0.01 ( e ) A physical interaction between WIPI2 and ATG101. HEK293T cells were co-transfected with GFP-WIPI2 and FLAG-ATG101 and incubated for 24–48 h in the treatment with vehicle or rapamycin (0.5 μM) for 4 h prior to harvesting. GFP-WIPI2 was immunoprecipitated with anti-GFP, then the immunoprecipitated complexes were analyzed for interaction between ATG101 and WIPI2 by western blotting using anti-FLAG. ( f ) Removal of the ATG101 C-terminus domain (ΔC) reduced the interaction between ATG101 and WIPI2. HEK293T cells were co-transfected with GFP-WIPI2 and FLAG-ATG101 FL or ΔC for 24–48 h and incubated for 4 h in complete medium (COM) or amino-acid deprived medium (–AA) for starvation. FLAG-ATG101 was immunoprecipitated with anti-FLAG, then immunoprecipitated, analyzed by western blotting using anti-WIPI2.

Article Snippet: Primary antibodies against ATG13 (13468), ATG101 (13492), LC3B (2775) and WIPI2 (8567) were purchased from Cell Signaling Technology (Danvers, MA, USA); Antibodies against HUWE1 (A300-486), β-actin (A300–491A), HA (A190–108A) and WIPI2 (A305-324A) were purchased from Bethyl Laboratories(Montgomery, TX, USA); those against FLAG M2 (F1804) and FLAG (F7425) were purchased from Sigma Aldrich (St. Louis, MO, USA); antibody against GFP (mouse SC-9996)(rabbit SC 8334) were purchased from Santa Cruz Biotechnology; and antibody against p62(610832) was purchased from BD Bioscience; antibody against ATG14 (GTX119950) was purchased from GeneTex (Hsinchu, Taiwan).

Techniques: Stable Transfection, Expressing, Incubation, Imaging, Activity Assay, Western Blot, Marker, Transfection, Staining, Immunoprecipitation

Journal: International Journal of Molecular Sciences

Article Title: ATG101 Degradation by HUWE1-Mediated Ubiquitination Impairs Autophagy and Reduces Survival in Cancer Cells

doi: 10.3390/ijms22179182

Figure Lengend Snippet: ATG101-mediated autophagy supported while HUWE1-catalyzed ubiquitination of ATG101 reduced cancer cell survival. ( a ) Knockdown of HUWE1 reduced the apoptotic death of cancer cells under nutrient starvation as assessed by Annexin V/propidium iodide (PI) staining and flow cytometry. shCTL or shHUWE1 MIA PaCa-2 cells were plated overnight and incubated in amino acid deprivation (–AA) medium for 48 h. Error bars indicate the mean ± SEM of three independent experiments. *** p < 0.001. ( b ) Additional knockdown of ATG101 and/or WIPI2 further enhanced cell death in HUWE1 knockdown cells. shCTL or shHUWE1 MIA PaCa-2 cells were transfected with WIPI2 and/or ATG101 siRNA for 24 h and subsequently incubated in HBSS for 24 h. Cell death was analyzed as in ( a ). Error bars indicate the mean ± SEM of three independent experiments. * p < 0.05; *** p < 0.001. ( c ) Schematic diagram of HUWE1 targeting ATG101 for ubiquitination and degradation, promoting cancer cell death via blocking autophagy activation under metabolic stress.

Article Snippet: Primary antibodies against ATG13 (13468), ATG101 (13492), LC3B (2775) and WIPI2 (8567) were purchased from Cell Signaling Technology (Danvers, MA, USA); Antibodies against HUWE1 (A300-486), β-actin (A300–491A), HA (A190–108A) and WIPI2 (A305-324A) were purchased from Bethyl Laboratories(Montgomery, TX, USA); those against FLAG M2 (F1804) and FLAG (F7425) were purchased from Sigma Aldrich (St. Louis, MO, USA); antibody against GFP (mouse SC-9996)(rabbit SC 8334) were purchased from Santa Cruz Biotechnology; and antibody against p62(610832) was purchased from BD Bioscience; antibody against ATG14 (GTX119950) was purchased from GeneTex (Hsinchu, Taiwan).

Techniques: Ubiquitin Proteomics, Knockdown, Staining, Flow Cytometry, Incubation, Transfection, Blocking Assay, Activation Assay