Journal: Autophagy

Article Title: Ambra1 haploinsufficiency in CD1 mice results in metabolic alterations and exacerbates age-associated retinal degeneration

doi: 10.1080/15548627.2022.2103307

Figure Lengend Snippet: Ambra1 +/gt retinas show a pronounced decrease in autophagy and increased protein aggregation. (A) Changes in autophagic flux determined by western blot for LC3-II in the absence or presence of NH 4 Cl and leupeptin (NL, to block lysosomal degradation) in whole retina extracts from young Ambra1 +/+ and Ambra1 +/gt mice. Quantification of autophagic flux in young (3 months), middle-aged (MA) (12–15 months), and old mice (22–26 months) is shown in the right-hand panel. Western blots corresponding to middle-aged and old mice are shown in Figure S1. Autophagic flux is determined as the ratio between NL and Control (n = 3–4 per group). (B) Decreased mRNA expression of Atg5, Sqstm1, Tfeb and Wipi2 as determined by qPCR of whole retina extracts from young, middle-aged, and old Ambra1 +/+ and Ambra1 +/gt mice (n = 3–4 per group). (C-F) Representative images and quantification of LC3 puncta in the outer nuclear layer (cyan) (C) and protein aggregates in the whole retina as measured by ProteoStat® protein aggregation assay (yellow) (D) in young, middle-aged, and old Ambra1 +/+ and Ambra1 +/gt mice. Nuclei were counterstained with DAPI (gray). Graphs show quantification of LC3 + puncta (E) and protein aggregates (F) (n = 3–9 per group). Data are presented as the mean ± SEM. *p < 0.05: two-tailed Student´s t -test or Mann-Whitney U -test (A, E, F); two-way ANOVA followed by post hoc LSD test for age (A and E) and genotype ( B ). Scale bars: 25 µm.

Article Snippet: The following TaqMan gene expression probes were used: Sqstm1 (Mm00448091_m1), Atg5 (Mm00504340_m1), Wipi2 (Mm00617842_m1), Tfeb (Mm00448968_m1), Ppargc1a (Mm01208835_m1), Nrf1 (Mm01135606_m1), Nfe2l2 (Mm00477784_m1), Tfam (Mm00447485_m1), Timm23 (Hs00197056_m1), Cox4i1 (Mm01250084_m1), Gapdh (Mm99999915_g1), Hk2 (Mm00443385_m1), Pkm (Mm00834102_gH), Nqo1 (Mm01253561_m1) and Pdha1 (Mm00468675_m1).

Techniques: Western Blot, Blocking Assay, Control, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Science Advances

Article Title: Restructuring of the plasma membrane upon damage by LC3-associated macropinocytosis

doi: 10.1126/sciadv.abg1969

Figure Lengend Snippet: Analysis of LC3 response in MCF7 eGFP-LC3 cells. ( A ) Representative images of cells transfected with Ctrl or ULK1 siRNA (72 hours) and exposed to rapamycin treatment (200 nM) or laser injury (white arrows). ( B ) Corresponding immunoblot of lysates after siRNA transfection (72 hours) (Hsp90, loading control). ( C ) Number of LC3 puncta per cell after treatment with rapamycin (200 nM) in cells transfected with Ctrl or ULK1 siRNA (72 hours). At least eight cells per experiment were analyzed from three experiments. Mean + SD. ( D ) Quantification of eGFP-LC3 fluorescence around the repair site following laser injury (black arrow) in cells transfected with ULK1 or Ctrl siRNA (72 hours). Mean ± SEM of normalized eGFP-LC3 intensity from eight cells per condition from three experiments. ( E ) Images of LC3 puncta formation after treatment with rapamycin (30 min) in cells transfected with Ctrl or ATG13 siRNA (72 hours) and ( F ) corresponding immunoblot after siRNA transfection (72 hours) (GAPDH, loading control). Extended blot is shown in fig. S5C. ( G ) Number of LC3 puncta per cell after rapamycin (30 min) in cells transfected with Ctrl or ATG13 siRNA (72 hours). Mean + SD, 12 cells per condition from three experiments. ( H ) LC3 puncta formation after laser injury (white arrows) in cells transfected with Ctrl or ATG13 siRNA (72 hours). ( I ) Quantification of eGFP-LC3 fluorescence around the repair site following laser injury (black arrow). Mean ± SEM of normalized intensity from nine cells per condition from three experiments. ( J ) Cells expressing RFP-WIPI2 before and after laser injury (white arrow). Blue arrows, eGFP-LC3–positive puncta. Yellow arrows, puncta positive for RFP-WIPI2 and eGFP-LC3. Individual channels are shown in fig. S6. ( K ) Sequential images of MCF7 eGFP-LC3 cells expressing mCherry-p62 and exposed to laser injury (white arrow). Yellow arrows, puncta positive for both mCherry-p62 and eGFP-LC3. Individual channels are shown in fig. S7. ( L ) Quantification of eGFP-LC3 puncta around the injury site (black arrow) in cells depleted for p62 by siRNA (72 hours). Mean ± SEM, from 14 cells per condition from three experiments. ( M ) Corresponding immunoblot from p62 siRNA transfection (72 hours) (Hsp90: loading control). Extended blot is shown in fig. S5B. ( N ) eGFP-LC3 puncta formation after laser injury (white arrow) in cells depleted for p62 as compared to Ctrl siRNA. Note that p62 depletion induces some nonspecific accumulation of eGFP-LC3 without injury. ( O ) MCF7 cells depleted for p62 by siRNA (72 hours) and exposed to glass bead injury (by vortex) and then reseeded and assayed for cell death (24 hours) by PI/Hoechst 33342 exclusion. Mean + SD, of five independent experiments. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. Two-way ANOVA with Bonferroni’s multiple comparisons test or unpaired t test of AUC values with Welsh’s correction.

Article Snippet: Expression plasmid with OFPSpark/RFP N-terminal tag containing human WIPI2 cDNA was purchased from Sino Biological Inc. (HG14898-ANR).

Techniques: Transfection, Western Blot, Fluorescence, Expressing

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: Nature Communications

Article Title: WIPI3 and WIPI4 β-propellers are scaffolds for LKB1-AMPK-TSC signalling circuits in the control of autophagy

doi: 10.1038/ncomms15637

Figure Lengend Snippet: ( a ) Structural homology modelling using HHpred. Propeller blades are indicated (1 to 7), and unstructured sequences are omitted. ( b ) Protein-phospholipid binding overlay assays using G361 cell extracts followed by the detection of endogenous WIPI1, WIPI2 or WIPI4 or using a monoclonal U2OS cell line stably expressing GFP-WIPI3 followed by anti-GFP enhanced chemiluminescence (ECL) detection (left panels). Monoclonal U2OS cell lines stably expressing GFP-WIPI1, GFP-WIPI2B or GFP-WIPI3 or transiently expressing GFP-WIPI4 were starved for 3 h with nutrient-free medium. Images were acquired by confocal LSM and processed using Volocity to generate 3D-reconstruction fly-through movies . Representative still images are presented (right panels). Scale bars: 3 μm. ( c ) U2OS cells stably expressing GFP-WIPI1, GFP-WIPI2B or GFP-WIPI3 or transiently expressing GFP-WIPI4 were fed (F) or starved (S) for 3 h with or without bafilomycin A1 (BafA1) or LY294002 (LY). The percentage GFP-WIPI puncta cells were calculated (up to 429 cells per condition, n =3). ( d ) U2OS cells stably expressing GFP-WIPI1, GFP-WIPI2B or GFP-WIPI3 or transiently expressing GFP-WIPI4 were starved (3 h), and endogenous ATG12, LC3, p62 (anti-ATG12, anti-LC3, anti-p62 and IgG-Alexa Fluor 546 antibodies) or transiently expressed myc-tagged ATG14 or DFCP1 detected (anti-myc/IgG-Alexa Fluor 546 antibodies). Merged confocal LSM images are presented (left panels, dashed lines: cell boundaries; right panels: magnified subsections). Scale bars: 20 μm. ( e ) Confocal LSM examinations of starved (3 h) U2OS cells stably expressing GFP-WIPI1 and immunostained with anti-WIPI2/IgG-Alexa Fluor 546 and anti-WIPI4/IgG-Alexa Fluor 633 antibodies. Intensity profiles of co-localizations (peaks 1 and 2, upper panel) are displayed, along with the corresponding magnified image sections. Scale bar: 2.5 μm. ( f ) U2OS cells stably expressing GFP-WIPI3 and transiently expressing myc-tagged WIPI1 or WIPI2B were starved (3 h) and immunostained using anti-myc or anti-WIPI4 and IgG-Alexa Fluor 546 antibodies. Scale bar: 2.5 μm. Arrows in magnified image sections indicate co-localization events ( d – f ). is available: , . Statistics and source data can be found in . Mean±s.d.; heteroscedastic t -testing; P values: * P <0.05, ** P <0.01, *** P <0.001, ns: not significant.

Article Snippet: Standard reverse transcription (RT)–PCR was performed with the following TaqMan probes (Applied Biosystems): Hs00215872_m1 (WIPI1), Hs00255379_m1 (WIPI2), Hs00750495_s1 (WIPI3/WDR45B), Hs01079049_g1 (WIPI4/WDR45), Hs00388292_m1 (NUAK2), Hs00908871_m1 (BRSK2).

Techniques: Binding Assay, Stable Transfection, Expressing

Journal: Nature Communications

Article Title: WIPI3 and WIPI4 β-propellers are scaffolds for LKB1-AMPK-TSC signalling circuits in the control of autophagy

doi: 10.1038/ncomms15637

Figure Lengend Snippet: G361 cell lines stably expressing shRNAs targeting WIPI1 (shWIPI1), WIPI2 (shWIPI2), WIPI3 (shWIPI3), WIPI4 (shWIPI4) or non-targeting shRNA (shControl) were assessed by quantitative RT–PCR ( a ) or electron microscopy analysis upon starvation (3 h), scale bars: 500 nm ( b ). ( c ) Monoclonal U2OS cell lines stably expressing GFP-LC3 and shWIPI2 or shControl were assessed by quantitative RT–PCR (left panel). Automated high-throughput image acquisition (middle panel, upper row: dashed lines indicate cell boundaries; lower row: magnified sections) and analysis (right panel). The numbers of GFP-LC3 puncta in control (shRNA) or WIPI2-KD (shWIPI2) cells were calculated under fed (F) or starved (S) conditions with or without wortmannin (WM) or bafilomycin A1 (BafA1). The mean number of GFP-LC3 puncta per cell was calculated (up to 15,401 cells per condition, n =3). Scale bars: 20 μm. ( d ) U2OS cells stably expressing GFP-LC3 were transiently transfected (48 h) with control siRNA (siControl) or siRNAs targeting WIPI3 (siWIPI3) or WIPI4 (siWIPI4). Total RNA was extracted for quantitative RT–PCR (left panel: WIPI3, middle panel: WIPI4). In addition, fed cells (F) cells were treated with or without of BafA1 for automated high-throughput image analysis (right panel). Mean numbers of GFP-LC3 puncta per cell are presented (up to 2,680 cells per condition, n =3). ( e ) Endogenous WIPI2 puncta formation was examined (anti-WIPI2/IgG-Alexa Fluor 488 antibodies) by confocal LSM and ImageJ (least 15 cells, n =3) using stable G361 WIPI3-KD (shWIPI3), WIPI4-KD (shWIPI4) and control cells (shControl). ( f ) G361 cells were transiently transfected with control siRNA (siControl), siWIPI3, siWIPI4 or a siWIPI3/siWIPI4 combination and fed (F) or starved (S) for 3 h with or without BafA1. Cell lysates were analysed by anti-LC3 and anti-tubulin western blotting. The migrations of LC3-I and LC3-II are indicated (left panel). LC3-I (middle panel) and LC3-II (right panel) abundances were quantified and results achieved in fed conditions (F) are presented ( n =3). is available: . Statistics and source data can be found in . Mean±s.d.; heteroscedastic t -testing; P values: * P <0.05, ** P <0.01, *** P <0.001, ns: not significant.

Article Snippet: Standard reverse transcription (RT)–PCR was performed with the following TaqMan probes (Applied Biosystems): Hs00215872_m1 (WIPI1), Hs00255379_m1 (WIPI2), Hs00750495_s1 (WIPI3/WDR45B), Hs01079049_g1 (WIPI4/WDR45), Hs00388292_m1 (NUAK2), Hs00908871_m1 (BRSK2).

Techniques: Stable Transfection, Expressing, shRNA, Quantitative RT-PCR, Electron Microscopy, High Throughput Screening Assay, Control, Transfection, Western Blot

Journal: Nature Communications

Article Title: WIPI3 and WIPI4 β-propellers are scaffolds for LKB1-AMPK-TSC signalling circuits in the control of autophagy

doi: 10.1038/ncomms15637

Figure Lengend Snippet: Monoclonal U2OS cell lines stably expressing GFP-WIPI1, GFP-WIPI2B, GFP-WIPI2D, GFP-WIPI3, GFP-WIPI4 or GFP alone were analysed by anti-GFP immunoprecipitation followed by nano liquid chromatography (LC)-MS/MS analysis (for detailed information, see Materials and Methods). ( a ) GeneMANIA was used to visualize identified WIPI protein–protein interactions. Red lines represent interactions identified in this study, and grey lines represent previously reported and predicted interactions (GeneMANIA, see Materials and Methods). ATG proteins and autophagy regulators are indicated with red letters. Complete results can be found in . Interactions identified using GFP-WIPI2B and GFP-WIPI2D were combined and are presented as WIPI2 interactions. Proteins exclusively interacting with either of the two WIPI2 isoforms can be found in . ( b ) A sub-network of the WIPI protein interactome focusing on autophagy-related and autophagy-relevant proteins is shown. Red lines represent interactions identified in this study, with dotted red lines representing interactions with low peptide counts in our LC-MS/MS analysis. Grey lines represent previously reported and predicted interactions (GeneMANIA, see Materials and Methods). In addition, the reported interactions of FIP200-ATG16L, TSC1-FIP200 and AMPK-TSC2 that did not appear when using GeneMANIA are also shown in grey. demonstrating WIPI1, WIPI2B, WIPI2D and WIPI4 co-immunoprecipitation with NudC is available.

Article Snippet: Standard reverse transcription (RT)–PCR was performed with the following TaqMan probes (Applied Biosystems): Hs00215872_m1 (WIPI1), Hs00255379_m1 (WIPI2), Hs00750495_s1 (WIPI3/WDR45B), Hs01079049_g1 (WIPI4/WDR45), Hs00388292_m1 (NUAK2), Hs00908871_m1 (BRSK2).

Techniques: Stable Transfection, Expressing, Immunoprecipitation, Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Protein-Protein interactions

Journal: Nature Communications

Article Title: WIPI3 and WIPI4 β-propellers are scaffolds for LKB1-AMPK-TSC signalling circuits in the control of autophagy

doi: 10.1038/ncomms15637

Figure Lengend Snippet: ( a ) U2OS cells transiently expressing GFP-WIPI1, GFP-WIPI2B, GFP-WIPI2D, GFP-WIPI3, GFP-WIPI4 or GFP alone were starved for 3 h and analysed by anti-GFP immunoprecipitation followed by anti-GFP or anti-ATG16L immunoblotting. The asterisk in the right panel (GFP-IP) represents a nonspecific band. Endogenous ATG16L isoforms co-precipitated with GFP-WIPI1, GFP-WIPI2B and GFP-WIPI2D as indicated in the right panel. ( b , c ) G361 cells stably expressing shRNA targeting WIPI2 ( b , shWIPI2), WIPI1 ( c , shWIPI1) or the non-targeting control (shControl) were fed (F) or starved (S) for 3 h, and endogenous ATG16L was detected using anti-ATG16L/IgG-Alexa Fluor 488 antibodies and fluorescence microscopy. Mean percentages of ATG16L-puncta-positive cells (up to 369 individual cells per condition, n =3) are presented. ( d ) Monoclonal U2OS cells stably expressing GFP-WIPI1 and shWIPI2 or shControl were starved (S) for 3 h, and the percentage of cells displaying an accumulation of perinuclear GFP-WIPI1 was calculated using fluorescence microscopy. Mean values (400 cells per condition, n =4) are presented. ( e , f ) G361 cells stably expressing shRNAs targeting WIPI1 ( e , shWIPI1) or WIPI2 ( f , shWIPI2) were fed (F) or starved (S) for 3 h and immunostained using anti-WIPI4/IgG-Alexa Fluor 488 antibodies. Mean percentages of WIPI4-puncta-positive cells (up to 340 cells per condition, n =3) are presented. ( g ) G361 cells stably expressing shRNAs targeting WIPI3 (shWIPI3), WIPI4 (shWIPI4) or the non-targeting shRNA control (shControl) were fed (F) or starved (S) for 3 h and immunostained using anti-ATG16L/IgG-Alexa Fluor 488 antibodies for analysis by fluorescence microscopy (left panel). Mean percentages of ATG16L-puncta-positive cells (up to 387 cells per condition, n =3) are presented (right panel). ( h , i ) ATG5 wild-type (WT) or knockout (KO) mouse embryonic fibroblasts (MEFs) transiently expressing GFP-WIPI1, GFP-WIPI2B, GFP-WIPI3 or GFP-WIPI4 were analysed by confocal LSM, and images from ATG5 WT MEFs ( h , upper panel) were processed using Volocity to generate 3D-reconstruction fly-through movies , from which still images are presented ( h , lower panel). Mean percentages of GFP-WIPI puncta-positive cells (300 cells, n =3). Mean±s.d.; heteroscedastic t -testing; P values: * P <0.05, ns: not significant. Scale bars: 20 μm. Statistics and source data can be found in .

Article Snippet: Standard reverse transcription (RT)–PCR was performed with the following TaqMan probes (Applied Biosystems): Hs00215872_m1 (WIPI1), Hs00255379_m1 (WIPI2), Hs00750495_s1 (WIPI3/WDR45B), Hs01079049_g1 (WIPI4/WDR45), Hs00388292_m1 (NUAK2), Hs00908871_m1 (BRSK2).

Techniques: Expressing, Immunoprecipitation, Western Blot, Stable Transfection, shRNA, Control, Fluorescence, Microscopy, Knock-Out

Journal: Nature Communications

Article Title: WIPI3 and WIPI4 β-propellers are scaffolds for LKB1-AMPK-TSC signalling circuits in the control of autophagy

doi: 10.1038/ncomms15637

Figure Lengend Snippet: ( a ) Stable GFP-WIPI1, GFP-WIPI2B, GFP-WIPI3 or GFP-WIPI4 U2OS cells were starved (3 h), and subjected to anti-GFP immunoprecipitation and anti-TSC2, anti-phospho-TSC2 (S1387), anti-TSC1 or anti-GFP immunoblotting. ( b ) U2OS cells were analysed by anti-TSC1 immunoprecipitation (TSC1-IP), anti-TSC1, anti-TSC2 and anti-WIPI1 (right panel), anti-WIPI2 (right panel), anti-WIPI3 (left panel) or anti-WIPI4 (right panel) immunoblotting. ( c ) ATG5 WT or KO MEFs expressing GFP-WIPI3 (W3) or GFP were subjected to anti-GFP immunoprecipitation and anti-TSC1, anti-TSC2 and anti-GFP immunoblotting. Conditions (3 h): fed (F) or starved (S). ( d ) Stable GFP-WIPI3 or GFP U2OS cells were analysed by anti-GFP immunoprecipitation and anti-TSC2 and anti-GFP immunoblotting. Conditions (15 min. to 3 h): fed (F), starved (S), starved with LY294002 (S+LY). ( e ) Myc-tagged TSC1 fragments (TSC1-M, TSC1-C; full length: TSC-FL) were expressed in stable GFP-WIPI3 U2OS cells and subjected to anti-GFP immunoprecipitation and anti-GFP or anti-myc immunoblotting. ( f ) Lysosomal fractions (no. 1–7; total protein control: TP) from stable GFP-WIPI3 U2OS cells (BafA1, 3 h) were immunoblotted using anti-GFP, anti-LAMP2, anti-TSC2, anti-WIPI2 or anti-Rab4 antibodies. ( g ) Stable GFP-WIPI3 U2OS cells were starved (3 h) and immunostained with anti-TSC2/IgG-Alexa Fluor 546 and anti-LAMP2/IgG-Alexa Fluor 633 antibodies for confocal LSM. ( h ) Stable GFP-WIPI3 U2OS cells were immunostained with anti-Lamp2/IgG-Alexa Fluor 633 and anti-WIPI2/IgG-Alexa Fluor 546 for confocal LSM. Conditions (3 h): fed (F), starved (S). ( i ) Stable GFP-WIPI3 U2OS cells with siControl or siTSC2 were fed (F) or starved (S) with or without BafA1 (3 h). Upper panel: anti-TSC2 immunoblotting, lower panel: GFP-WIPI3 puncta assessment (up to 493 cells per condition, n =4). ( j ) U2OS cells were subjected to immunoblotting using anti-phospho-mTOR (S2481), anti-mTOR, anti-phospho-ULK1 (S757), anti-ULK1 and anti-tubulin antibodies. Treatments: fed (F, 4 h), starved (S, 4 h), starved (3 h) and fed (1 h) (S→F). ( k ) Stable GFP-WIPI3 U2OS cells were immunostained with anti-mTOR/IgG-Alexa Fluor 546 and anti-LAMP2/IgG-Alexa Fluor 633 antibodies. Treatments: starved (S, 2 h), starved (2 h) and fed (1 h) (S→F). Co-localizations are indicated with arrows ( g , h , k ). is available: . Statistics and source data: . Mean±s.d.; heteroscedastic t -testing; P values: * P <0.05, ** P <0.01, *** P <0.001. Scale bars: 3 μm.

Article Snippet: Standard reverse transcription (RT)–PCR was performed with the following TaqMan probes (Applied Biosystems): Hs00215872_m1 (WIPI1), Hs00255379_m1 (WIPI2), Hs00750495_s1 (WIPI3/WDR45B), Hs01079049_g1 (WIPI4/WDR45), Hs00388292_m1 (NUAK2), Hs00908871_m1 (BRSK2).

Techniques: Immunoprecipitation, Western Blot, Expressing, 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

Journal: PLoS ONE

Article Title: DNA methylation in blood—Potential to provide new insights into cell biology

doi: 10.1371/journal.pone.0241367

Figure Lengend Snippet: Annotation, methylation status and TaqMan probe information for the 11 selected CpG sites.

Article Snippet: cg02665297 , CD19 , 0.08 , 0.95 , 0.87 , 86.91 , 7 , 5270984 , WIPI2 , 3’UTR , Hs01093807_m1.

Techniques: Methylation