EMBO/soda-nel huggingface dataset:
ULK1 induces autophagy by phosphorylating Beclin-1 and activating VPS34 lipid kinase (2013)
figf6

Unless otherwise stated, all experiments were repeated three times and data shown are representative. (a) HEK293 cells were transfected with the indicated plasmids. Cells were grown in the presence of amino acids and treated with NH4Cl to block autophagic turnover where indicated. Lysates were immunoblotted with the indicated antibodies. (b) HEK293 cells transfected with Beclin-1 ATG14L were grown in the presence or absence of amino acids. Lysates were immunoblotted with the indicated antibodies (left panel) and quantified by densitometry (right panel). Data represent mean+s.d. of three unique experiments. (c) Beclin-1-shRNA reconstituted lines (WT or mutant) and controls (scramble shRNA or Beclin-1 shRNA) were grown with or without amino acids and assessed for autophagy (left panel) and Beclin-1 levels (right panel). (d) Autophagosome (denoted by arrowheads) generation on amino-acid withdrawal was analysed by electron microscopy. Cell lines and conditions from c were used and representative images from the indicated amino-acid-starved lines are shown; scale bars, 0.4 μm. (e) Quantification of d. Fold induction was determined by arbitrarily making the nutrient-rich condition 1 (solid bars) for each line. Error bars represent the s.d. of the mean value over an average of 20 fields of view within a representative experiment. NS, not significant. (f) HA–Beclin-1 WT or S14D was transiently expressed in FIP200−/− MEFs grown under nutrient-rich conditions. Indirect immunofluorescence was performed using antibodies against endogenous LC3B and HA–Beclin-1. Scale bars, 20 μm. (g) Quantification of LC3B puncta from confocal in f. In the HA–Beclin-1- or HA–Beclin-1-S14D-transfected samples, only the HA-positive cells were counted for LC3B puncta. Error bars were processed as in e. Mean value shown;
P values determined by Student’s
t-test using 10 unique fields of view from f. Uncropped images of blots are shown in Supplementary Fig. S4.
ULK1 induces autophagy by phosphorylating Beclin-1 and activating VPS34 lipid kinase (2013)
figf7

Unless otherwise stated, all experiments were repeated three times and data shown are representative. (a) Bec-1 C. elegans were reconstituted with either WT or mutant GFP–BEC-1. Stable worm lines with Bec-1 rescue were obtained and embryos were stained with anti- PGL-1 antibody. Arrowheads indicate normal PGL-1 staining in germline cells. (b) Quantification of PGL-1 puncta outside germline cells (left panel). Error bars represents s.d. between 3 unique embryos in a representative experiment. Reconstituted Bec-1 (WT and mutant) levels in Bec−/− stable worms were compared by western blotting. Mean value presented. (c) Spectrum of defects in PGL granule degradation in bec-1 mutant rescue embryos. Mutant embryos exhibited either high levels of diffuse PGL-1 staining (middle-left panel; one-third of the embryos), or large punctuate PGL-1 structures in somatic cells (bottom-left panel; two-thirds of the embryos). Both diffuse or punctuate PGL-1 staining in somatic cells have been described in autophagy-deficient embryos. (d) Embryos from the lines described in a were labelled with anti-LGG-1, along with WT and unc-51 worms. Representative embryos at ∼ 100 cell stage are shown. (e) Quantification of LGG-1 per embryo from labelling in d. Error bars generated as in b. Mean value presented. Uncropped images of blots are shown in Supplementary Fig. S4. Scale bar, 10 μm.
Two newly identified sites in the ubiquitin‐like protein Atg8 are essential for autophagy (2006)
Figure 1

Modelling the three‐dimensional structure of Atg8. (A) Multiple sequence alignment of Atg8 homologues. Amino‐acid sequences of Atg8 and its mammalian homologues are aligned using the Clustal_W program. Underlined residues represent mutated amino acids in Atg8. (B) Alignment of Atg8 predicted three‐dimensional structure with its mammalian homologues. SWISS MODEL was used to predict Atg8 structure and PyMol was used for presentation. Orange represents the predicted Atg8 structure, and red, blue and green represent GATE‐16, GABARAP and LC3, respectively. (C) Three‐dimensional model of Atg8. The predicted structure contains a ubiquitin fold decorated by two further amino‐terminal helices (amino acids Tyr 49, Leu 50, Phe 77 and Phe 79 are marked in green).
Two newly identified sites in the ubiquitin‐like protein Atg8 are essential for autophagy (2006)
Figure 2

GFP–Atg8F77/79A double mutant shows a defect in the autophagic process. GFP–Atg8WT, GFP–Atg8F77A, GFP–Atg8F79A or GFP–Atg8F77/79A was transformed into Δatg8 cells. (A) Cells were grown to mid‐exponential phase and then incubated in the presence of 3.5% galactose to induce expression of GFP–Atg8. Aminopeptidase I (API) maturation was tested in cells incubated in control or starvation medium by western blot analysis using anti‐Atg8 (right bottom panel), anti‐GFP (left bottom panel) or anti‐API antibodies (upper panel). (B) Cells were pulse‐labelled with [35S]methionine and chased on non‐radioactive starvation medium. Aliquots were taken at the indicated times and acid‐soluble small peptides generated by proteolysis were determined. (C) Cells were grown in control or in starvation medium and visualized by light microscopy. Arrows point to the pre‐autophagosomal structure (PAS), and arrowheads point to GFP within the vacuoles. (D) Cell extracts were subjected to SDS–polyacrylamide gel electrophoresis followed by immunoblot analysis using anti‐GFP antibodies. GFP, green fluorescent protein; mAPI, mature aminopeptidase I; pAPI, premature aminopeptidase I; WT, wild type.
Two newly identified sites in the ubiquitin‐like protein Atg8 are essential for autophagy (2006)
Figure 3

Atg8F77/79A does not undergo cleavage in the carboxyl terminus. (A) Atg8WT–HA, Atg8F77A–HA, Atg8F79A–HA or Atg8F77/79A–HA was transformed into Δatg8 cells. Cell extracts were prepared and subjected to 13.5% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) containing 6 M urea, followed by immunoblot analysis with anti‐Atg8 antibodies. (B) Prediction of protein–protein interaction sites in Atg8 (Neuvirth et al, 2004). A three‐dimensional model of Atg8 is coloured according to ProMate prediction (see supplementary information online) with red representing the predicted protein–protein binding sites, and Phe 77 and Phe 79 are marked in green within the prediction site. (C) Pull‐down assay of Atg8 mutants with Atg4. His–Atg4 was attached to cobalt beads (Talon metal‐affinity resin), washed and incubated with total cell extracts of Atg8 mutants. Elution was subjected to SDS–PAGE followed by western blot analysis using anti‐Atg8 and anti‐Atg4 antibodies. (D) Localization of green fluorescent protein (GFP)–Atg8wt and GFP–Atg877/79 cells carrying Atg4 plasmid under normal (control) and starvation conditions. Arrows point to the pre‐autophagosomal structure, and arrowheads point to GFP within the vacuoles. (E) Aminopeptidase I (API) maturation was tested in cells incubated in control or starvation medium by western blot analysis using anti‐API antibodies. mAPI, mature aminopeptidase I; pAPI, premature aminopeptidase I; WT, wild type.
Two newly identified sites in the ubiquitin‐like protein Atg8 are essential for autophagy (2006)
Figure 4

GFP–Atg8F77/79AΔR double mutant shows a defect in the autophagic process despite undergoing lipidation. GFP–Atg8WTΔR or GFP–Atg8F77/79AΔR was transformed into ΔAtg8 cells. (A) Cell extracts were subjected to 13.5% SDS–polyacrylamide gel electrophoresis containing 6 M urea, followed by immunoblot analysis using anti‐Atg8‐15N peptide antibodies. (B) Cells were grown in control or in starvation medium and visualized using fluorescence microscopy. Arrows point to the pre‐autophagosomal structure, whereas arrowheads point to GFP within the vacuoles. (C) Aminopeptidase I (API) maturation and GFP cleavage were tested as described above. Asterisk represents nonspecific band. GFP, green fluorescent protein; mAPI, mature aminopeptidase I; pAPI, premature aminopeptidase I; WT, wild type.
Two newly identified sites in the ubiquitin‐like protein Atg8 are essential for autophagy (2006)
Figure 5

GFP–Atg8L50A or GFP–Atg8Y49A transformed into Δatg8 cells does not reconstitute. (A) Δatg8 cells transformed with GFP–Atg8WT, GFP–Atg8L50A or GFP–Atg8Y49A constructs were grown to mid‐exponential phase and then incubated in the presence of 3.5% galactose to induce expression of GFP–Atg8. API maturation was tested using anti‐Atg8 (right bottom panel), anti‐GFP (left bottom panel) or anti‐API antibodies. (B) Starvation‐induced protein degradation of Δatg8 harbouring the different mutants was tested as described above. API, aminopeptidase I; GFP, green fluorescent protein; mAPI, mature aminopeptidase I; pAPI, premature aminopeptidase I; WT, wild type.
Two newly identified sites in the ubiquitin‐like protein Atg8 are essential for autophagy (2006)
Figure 6

GFP–Atg8 mutants were tested for their carboxy‐terminus processing, phosphatidylethanolamine conjugation and autophagic activity. Cells expressing GFP–Atg8WT or GFP–Atg8 mutants were grown in control or starvation medium. (A) GFP–Atg8 localization was determined using fluorescence microscopy (arrows depict pre‐autophagosomal structure, and arrowheads point to GFP within vacuoles). Cells were also tested for the appearance of autophagic bodies by using light microscopy. (B) Immunoblot analysis of different cell extracts was carried out using anti‐GFP antibodies (asterisk represents nonspecific band). (C) Atg8WT–HA, Atg8L50A–HA or Atg8Y49A–HA was transformed into Δatg8 and Δatg8Δatg4 double knockout strains and cell extracts were subjected to SDS–polyacrylamide gel electrophoresis (SDS–PAGE) followed by immunoblot analysis using anti‐HA antibodies (lower panel; asterisk represents nonspecific band). (D) Atg8WT, Atg8L50A or Atg8Y49A was transformed into Δatg8 and Δatg8Δatg4 double knockout strains and grown under control or starvation conditions for 4 h. Cell extracts were subjected to 13.5% SDS–PAGE containing 6 M urea, followed by immunoblot analysis using anti‐Atg8‐N15 antibodies. GFP, green fluorescent protein; WT, wild type.
Fission yeast Cactin restricts telomere transcription and elongation by controlling Rap1 levels (2014)
Figure 1

Characterization of UP‐TERRA mutants

Telomeric lncRNA levels in the indicated deletion strains as analyzed by dot blot (top) or Northern blot hybridization (bottom) using a double‐stranded telomeric probe. Telomeric dot blot signals are expressed as fold increase relative to wtA after normalization through U6 snRNA. Bars and error bars are averages and s.d. from 4 independent experiments. U6 and 18S rRNA are shown as loading controls for Northern blotting. The asterisk indicates unspliced U6. Molecular weights are on the left in kilobases.
ARRET and αARRET Northern blot analysis of the indicated strains using strand‐specific probes.
Telomere length analysis of ApaI‐digested DNA from the indicated strains.
Quantification of subtelomeric DNA from ChIP experiments using strains carrying the indicated Myc‐tagged proteins (2A9 and 8B7: SPBC2A9.02‐Myc and SPBP8B7.08c‐Myc, respectively). Immunoprecipitated DNA is expressed as fraction of input DNA. An untagged strain (−) served as a negative control. Bars and error bars are averages and s.d. from three independent experiments. Statistical significance was assayed using the unpaired, two‐tailed Student's t‐test. ***P 0.001 relative to untagged strain.
Fission yeast Cactin restricts telomere transcription and elongation by controlling Rap1 levels (2014)
Figure 2

cay1∆ cells accumulate Tf2 LTR retrotransposon transcripts

Chromosome maps of differentially expressed genes in cay1∆ cells shown as fold enrichment over wt. Red dots are overexpressed genes and Tf2 retrotransposons, blue dots are overexpressed LTR sequences.
Northern blot analysis of tlh1/2+, Tf2 retrotransposons, and 18S rRNA (loading control) in wt, cay1∆, and trichostatin A (TSA)‐treated wt cells. Black arrowhead indicates tlh1/2+ mRNA; red arrowhead unprocessed Tf2 transcripts; and blue arrowhead processed Tf2 transcripts.
ChIP analysis of Cay1‐Myc binding to the indicated genomic loci. subtel: subtelomeres; Tf2: Tf2 retrotransposon genes; LTR: Tf2 LTR sequences; Tlh: tlh1/2+ genes; cen: centromeres. Immunoprecipitated DNA is normalized to input DNA and expressed as fold increase over untagged strains (unt).
ChIP analysis of H3K9ac, H3K9me3, and total H3 at the indicated genomic loci. Immunoprecipitated DNA is normalized to input DNA and expressed as fold increase over wt after subtraction of values obtained for negative control immunoprecipitations performed with beads only.

Data information: Bars and error bars are averages and s.d. from at least 4 (C) or 8 (D) independent experiments. Statistical significance was assayed using the unpaired, two‐tailed Student's t‐test. *P 0.05, **P 0.01, ***P 0.001 relative to wt.
Fission yeast Cactin restricts telomere transcription and elongation by controlling Rap1 levels (2014)
Figure 3

Rap1 pre‐mRNA splicing and protein stability are impaired in cay1∆ cells

Western blot analysis of endogenous Rap1 and Taz1 in the indicated strains. Act1 was used as a loading control. Asterisks indicate cross‐reacting bands.
RT–PCR analysis of splicing efficiencies of the indicated pre‐mRNAs in wt and cay1∆ cells. The prp1‐1 strain carries a thermosensitive allele of the splicing factor Prp1 and was used as a positive control for splicing impairment. prp1‐1 cells were grown at 36°C for 4 h before harvesting. Amplification products corresponding to unspliced and spliced mRNAs are indicated by asterisks and gene names, respectively. rap1T indicates amplification products obtained with oligonucleotides spanning rap1+ exon 3 and thereby amplifying both spliced and unspliced mRNA. Intronless act1+ was used as a loading control. Numbers at the bottom of each panel are ratios between spliced and unspliced forms (s/u) and spliced and act1+ (s/act1). Values are expressed as fold increase over wt.
Western blot analysis of Rap1‐YFP protein levels in the indicated strains upon nmt1 promoter shutoff by thiamine addition. Membranes were probed with antibodies against GFP and Act1 (loading control).
Quantification of Rap1‐YFP protein levels in experiments as in (C). Rap1‐YFP levels are expressed as fold increase over time 0 after normalization through Act1. Data points and error bars are averages and s.d. from at least four independent experiments. Statistical significance was assayed using the unpaired, two‐tailed Student's t‐test. *P 0.05, **P 0.01 for cay1+ versus cay1∆ samples.
Fission yeast Cactin restricts telomere transcription and elongation by controlling Rap1 levels (2014)
Figure 4

Cay1 and Rap1 genetically interact to maintain telomere homeostasis

Telomere length analysis of ApaI‐digested DNA from cay1∆trt1∆ cells harvested at increasing generation doublings (gen).
Telomere length analysis of ApaI‐digested DNA from the indicated strains. dh: centromeric dh repeats shown as loading control.
PFGE analysis of telomeric fusions in strains grown to logarithmic phase (log) or G1‐arrested by nitrogen starvation (G1). Genomic DNA was digested with NotI and hybridized to C, I, L, and M probes detecting terminal fragments of chromosomes I and II. Bands corresponding to chromosome end fusions are indicated (fused).
Northern blot analysis of ARIA, ARRET, Tf2 retrotransposons, and 18S rRNA (loading control) in the indicated strains.
qRT–PCR quantification of TERRA levels expressed as fold increase over wt after normalization through act1+ mRNA. Bars and error bars are averages and s.d. from 3 independent experiments. Statistical significance was assayed using the unpaired, two‐tailed Student's t‐test. **P 0.01 relative to wt.
ChIP analysis of H3K9ac and total H3 for the indicated strains. Immunoprecipitated DNA is normalized to input DNA and expressed as fold increase over wt after subtraction of values obtained for negative control immunoprecipitations performed with beads only. Bars and error bars are averages and s.d. from at least four independent experiments. Statistical significance was assayed using the unpaired, two‐tailed Student's t‐test. ***P 0.001 relative to wt.
Fission yeast Cactin restricts telomere transcription and elongation by controlling Rap1 levels (2014)
Figure 5

Proliferation of cay1∆ cells

Serial dilutions of the indicated strains were spotted on complete medium and grown at 30°C or 20°C. Otrt1∆ and Otrt1∆cay1∆ are strains with circularized chromosomes lacking telomeric sequence (Supplementary Fig S7).
Representative images of wt and cay1∆ cells grown for 24 h at 30°C or 20°C. Scale bar, 20 μm.
Quantification of cell length for strains grown in complete medium for 24 h at 30°C or 20°C. Each dot represents a single cell and median cell length (m) is indicated for each sample. Data are from three independent experiments and at least 100 cells were scored per sample in each experiment.
Top: Examples of DAPI‐stained wt and cay1∆ cells grown in complete medium for 24 h at 30°C or 20°C. The white arrowhead indicates a chromosome bridge. Scale bar, 10 μm. Bottom: Quantification of chromosome bridges. Bars and error bars are averages and s.d. from three independent experiments and at least 200 cells were scored per sample in each experiment. Statistical significance was assayed using the unpaired, two‐tailed Student's t‐test. ***P 0.001 relative to 30°C.
Fission yeast Cactin restricts telomere transcription and elongation by controlling Rap1 levels (2014)
Figure 6

Overexpression of Rap1‐YFP in cay1∆ cells

RT–PCR analysis of pre‐mRNA splicing efficiencies of rap1+ and poz1+ pre‐mRNAs in cay1+ and cay1∆ cells expressing Cay1‐YFP or Rap1‐YFP. Amplification products corresponding to unspliced and spliced mRNAs are indicated by asterisks and gene names, respectively. rap1T indicates amplification products obtained with oligonucleotides spanning rap1 exon 3 and thereby amplifying both spliced and unspliced mRNA. Intronless act1+ was used as a loading control. Numbers at the bottom of each panel are ratios between spliced and unspliced forms (s/u) and spliced and act1+ (s/act1). Values are expressed as fold increase over wt.
Western blot analysis of total proteins from early generation cay1+ and cay1∆ cells expressing Cay1‐YFP, Rap1‐YFP, and Taz1‐YFP using antibodies against Rap1, Taz1, and Act1 (loading control). Numbers at the bottom are ratios between Rap1 proteins (endogenous plus ectopic) and Act1 (Rap1/Act1). Values are expressed as fold increase over wt.
Left: Representative YFP fluorescence and DIC images of cay1∆ strains expressing Cay1‐YFP. Scale bar, 10 μm. Right: Representative YFP and RFP fluorescence images of Cay1‐YFP‐ and Pot1‐RFP‐expressing cells. Arrows point to YFP and RFP foci within the same nucleus demonstrating lack of co‐localization. Scale bar, 5 μm.
Representative YFP fluorescence and DIC images of cay1+ and cay1∆ strains expressing Rap1‐YFP. Scale bar, 5 μm.
Fission yeast Cactin restricts telomere transcription and elongation by controlling Rap1 levels (2014)
Figure 7

Rap1 ectopic expression reverts the telomeric defects of cay1∆ cells

Telomere length analysis of ApaI‐digested DNA from cells harvested at early (left) and late generations (right) after expression of YFP‐tagged proteins.
Northern blot analysis of ARIA, ARRET, and 18S rRNA (loading control) at early generations after expression of YFP‐tagged proteins. Numbers at the bottom are ratios between ARRET and 18S rRNA signals, expressed as fold increase over wt.
ChIP analysis of subtelomeric H3K9ac, H3K9me3, and total H3 in the indicated strains at early generations. Immunoprecipitated DNA is normalized to input DNA and expressed as fold increase over wt after subtraction of values obtained for negative control immunoprecipitations performed with beads only. Bars and error bars are averages and s.d. from 3 independent experiments. Statistical significance was assayed using the unpaired, two‐tailed Student's t‐test. *P 0.05, **P 0.01, ***P 0.001 relative to wt.
Serial dilutions of early generation cay1+ and cay1∆ cells expressing Cay1‐YFP and Rap1‐YFP were spotted on complete medium and grown at 30°C and 20°C.
Quantification of cell length in strains grown for 24 h in complete medium at 30°C or 20°C. Each dot represents a single cell and median cell length (m) is indicated for each sample. Data are from 3 independent experiments and at least 100 cells were scored per sample in each experiment.
Quantification of chromosome bridges of strains grown for 24 h in complete medium at 30°C or 20°C. Bars and error bars are averages and s.d. from three independent experiments and at least 200 cells were scored per sample in each experiment.
Fission yeast Cactin restricts telomere transcription and elongation by controlling Rap1 levels (2014)
Figure 8

Unprocessed Tf2 transcripts accumulate in pre‐mRNA splicing mutants

Northern blot analysis of Tf2 retrotransposons, U6 snRNA, and act1+ mRNA (loading control) in wt, cay1∆, and prp1‐1 cells. prp1‐1 cells were grown at 36°C for 4 h before harvesting. Black arrowhead indicates unprocessed Tf2 transcripts. The asterisk indicates unspliced U6. Numbers at the bottom are the ratios between Tf2 and act1+ signals (Tf2/Act1) and expressed as fold increase over wt.
Northern blot analysis as in (A) in mpn1+ and mpn1∆ strains carrying a stably integrated extra copy of the snu6+ gene or empty vector control plasmids (ev).
Unfolded Protein Response-Induced ERdj3 Secretion Links ER Stress to Extracellular Proteostasis (2015)
Figure 1

Figure 1. ER stress increases ERdj3 secretion

Immunoblot of media and lysate collected from HEK293T cells overexpressing ERdj3WT or ERdj3KDEL. Fresh media was conditioned on cells for 24 h prior to harvest.
qPCR analysis of ERdj3 mRNA in HEK293T cells treated with or without thapsigargin (Tg; 6 h, 100 nM). Error bars represent the mean ± 95% confidence interval as calculated in DataAssist 2.0 (n = 3).
Representative immunoblot of lysates and media collected from HEK293T-Rex cells stably expressing non-silencing (NS) or ERdj3 shRNA. Cells were treated with the indicated concentration of Tg in fresh media for 16 h.
Quantification of the fold increase of ERdj3 in lysates and media collected from HEK293T-Rex cells treated with the indicated concentration of Tg in fresh media for 16 h as in Figure 1C. *indicates a pvalue < 0.05; (n = 3).
Representative autoradiogram of [35S]-labeled ERdj3 in lysates and media collected from Huh7 cells incubated in the presence or absence of Tg (1 µM). The experimental paradigm for labeling is shown above.
Quantification of fraction intracellular (dark grey) and extracellular (light grey) [35S]-labeled ERdj3 following a 4 h chase relative to total [35S]-labeled ERdj3 at 4 h (as shown in Figure 1E). Error bars represent standard error from biological replicates (n = 3).
Quantification of total [35S]-labeled ERdj3 in media collected from cells pretreated with vehicle or Tg, as shown in Figure 1E. The media [35S]-labeled ERdj3 was measured by densitometry and is normalized to the amount of media [35S]-labeled ERdj3 at 4 h from vehicle-treated cells. Error bars represent standard error from biological replicates. *indicates a pvalue < 0.05; (n = 3).
Representative immunoblot and quantification of sera collected at the indicated times from DBA/2J mice fed a high fat (60% fat) diet after a 18 h fast. A schematic of the fasting/refeeding experimental setup is shown above. The quantification was normalized for each mouse to the amount of ERdj3 in the serum draw immediately prior the fast. *indicates a p-value < 0.05; (n = 6).