EMBO/soda-nel huggingface dataset:
Novel targets for Huntington's disease in an mTOR-independent autophagy pathway (2008)
figf7

(a) The proportions of EGFP-positive COS-7 cells with aggregates or cell death, after transfection with EGFP-HDQ74 and rheb or pcDNA3.1 (empty vector) (1:3 ratio) for 4 h and treatment with or without 10 μM calpastatin or 50 μM calpeptin for 48 h post-transfection. The control condition for assessing the effect of calpain inhibitors in rheb-transfected cells was taken as 1. Error bars show 95% confidence interval. (b) The proportions of EGFP-positive COS-7 cells with >5 EGFP-LC3 vesicles, transfected with EGFP-LC3 and pcDNA3.1 (empty vector) or rheb (1:3 ratio) for 4 h and treated with or without 10 μM calpastatin or 50 μM calpeptin for 24 h post-transfection. Error bars show 95% confidence interval. (c) The proportions of EGFP-positive COS-7 cells with aggregates, transfected with EGFP-HDQ74 and constitutively active (CA) m-calpain or pcDNA3.1 (empty vector) (1:3 ratio) for 4 h and treated with or without 0.2 μM rapamycin for 48 h post-transfection. Error bars show 95% confidence interval. (d) The proportions of EGFP-positive COS-7 cells with >5 EGFP-LC3 vesicles, transfected with EGFP-LC3 and CA m-calpain or pcDNA3.1 (empty vector) (1:3 ratio) for 4 h and treated with or without 0.2 μM rapamycin for 24 h post-transfection. Error bars show 95% confidence interval. (e,f) The proportions of EGFP-positive COS-7 cells with aggregates (e) and cell death (f) as in Figure 1c, treated with or without 0.2 μM rapamycin, 10 μM calpastatin or both for 48 h. Error bars show 95% confidence interval. (g) Clearance of soluble EGFP-HDQ74 in stable PC12 cells as in Figure 1b, treated with or without 0.2 μM rapamycin, 10 μM calpastatin or both for the 48 h switch-off period. Error bars show s.e.m. (h) Clearance of A53T α-synuclein in stable PC12 cells as in Figure 1a, treated with or without 0.2 μM rapamycin, 10 μM calpastatin or both for the 8 h switch-off period. Error bars show s.e.m. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.
Novel targets for Huntington's disease in an mTOR-independent autophagy pathway (2008)
figf8

(a) Western blot showing EGFP-HDQ71–induced degeneration of rod photoreceptors at 9 d.p.f. as indicated by the decrease in the abundance of of monomeric (∼36 kDa) and dimeric (∼72 kDa) rhodopsin. Rhodopsin expression is further reduced by 3 μM (±)-Bay K8644, and recovered upon treatment with 3 μM verapamil, 100 μM 2′5′ddA, 3 μM clonidine and 1 μM calpastatin. No general toxicity to the zebrafish at these concentrations was observed. (b) Expression of EGFP-HDQ71 significantly reduces the levels of rhodopsin expression when compared to wild-type (WT) nontransgenic controls. This effect is further abrogated by 3 μM (±)-Bay K8644, whereas 3 μM verapamil, 100 μM 2′5′ddA, 3 μM clonidine and 1 μM calpastatin all protected against rod photoreceptor degeneration. Error bars show s.e.m. (c) Magnified bright field and EGFP images of zebrafish eye sections, taken at the ventral marginal zone for each of the indicated treatments (with concentrations as in b), are shown. Scale bar, 10 μm. ***P 0.001; **P 0.01; *P 0.05. (d) The total number of aggregates across 8 × 12 μm sections was counted and compared to the EGFP-HDQ71 DMSO-treated control. 3 μM (±)-Bay K8644 was found to significantly increase the aggregate burden, whereas 3 μM verapamil, 1 μM calpastatin, 3 μM clonidine and 100 μM 2′5′ddA decreased the aggregate load. As in our cell models, these aggregates were insoluble in 4% triton/SDS. Error bars show s.e.m. ***P 0.001; **P 0.01; *P 0.05. (e) Schematic representation of an mTOR-independent autophagy pathway involving cAMP-Ca2+-calpains-Gsα with multiple drug targets. Intracellular cAMP levels are increased by adenylyl cyclase (AC) activity, thereby activating Epac, which subsequently activates Rap2B, a small G protein that activates PLC-ε, resulting in the production of IP3 and consequent Ca2+ release from the endoplasmic reticulum (ER). Intracellular Ca2+ levels are also increased by L-type Ca2+ channel agonists. An increase in intracytosolic Ca2+ activates calpains, which cleave and activate Gsα, which activates AC to increase cAMP levels, thereby forming a loop. Activation of this pathway inhibits autophagy. Multiple drug targets acting at various places in this pathway induce autophagy, including the following: I1R agonists (clonidine and rilmenidine) and the AC inhibitor 2′5′ddA, which decrease cAMP levels, agents lowering inositol and IP3 levels (carbamazepine (CBZ) and valproic acid (VPA)), L-type Ca2+ channel antagonists (verapamil and loperamide), calpain inhibitors (calpastatin and calpeptin) and the Gsα inhibitor NF449.
eIF4A inactivates TORC1 in response to amino acid starvation (2016)
Figure 1

Figure 1: eIF4A is required for complete TORC1 inactivation upon amino acid removal

(a-b) eIF4A knockdown in either S2 (a) or Kc167 (b) cells blunts the inactivation of TORC1 upon amino acid removal. Cells were treated with dsRNA targeting GFP as a negative control, or three independent, non-overlapping dsRNAs targeting eIF4A for 5 days and then incubated with complete medium or medium lacking only amino acids for 30 minutes. Representative of 3 biological replicates.

(c) Only knockdown of eIF4A, but not of other genes involved in translation, causes elevated TORC1 upon amino acid withdrawal. S2 cells were treated with dsRNAs for 5 days and then incubated with medium containing or lacking amino acids for 30 minutes. After cell lysis, levels of S6K phosphorylation (T398) were measured by dot blot analysis and normalized to total S6K levels. Results are summarized here for all genes involved in translation, and provided in Dataset EV1.

(d) Impaired inactivation of TORC1 in response to eIF4A knockdown is most apparent upon partial depletion of amino acids, caused by removal of amino acid subsets. After 5 days of knockdown, S2 cells were treated for 30 min with either complete Schneider’s medium (+aa), Schneider’s medium lacking all amino acids (-aa) or various subsets of amino acids, as indicated (where EAA “essential amino acids” = H,I,L,K,M,T,W,V). Error bars: std dev. n=3 biological replicates.

(e) Time course of amino-acid removal reveals that eIF4A knockdown Kc167 cells maintain elevated S6K phosphorylation up to the maximum possible timepoint of 60 minutes when the cells start dying (see drop in S6K and tubulin levels). Representative of two biological replicates.

(f) eIF4A mutant larvae have impaired TORC1 inactivation upon shifting to food lacking amino acids. Control (w1118) or eIF4A1006/1013 1st instar larvae were transferred from standard food to plates containing either standard fly food or PBS/1% agarose+2%sucrose for 1h prior to lysis and immunoblot analysis. Two biological replicates are shown. Representative of 3 biological replicates.
eIF4A inactivates TORC1 in response to amino acid starvation (2016)
Figure 2

Figure 2: Impaired TORC1 inactivation upon amino acid removal is not a general consequence of impaired translation

(a-a’) Although knockdown of eIF4A or eIF3-S2 equally blunt translation of EGFP from an inducible plasmid (a), only knockdown of eIF4A but not eIF3-S2 impairs TORC1 inactivation upon amino acid removal (a’). (a) S2 cells, treated with indicated dsRNAs for 5 days, on day 3 transfected with an inducible EGFP plasmid (pMT-EGFP) and on day 4 induced for 18h. Non-targeting LacZ dsRNA used as a negative control. (a’) S2 cells treated with indicated dsRNAs for 5 days, then incubated with medium lacking amino acids for indicated time prior to lysis. Representative of two biological replicates.

(b) Knockdown of eIF4A, but not other translation initiation factors, blunts TORC1 inactivation upon amino acid withdrawal. Kc167 cells treated with indicated dsRNAs for 4 days and then incubated with complete Schneider’s medium or Schneider’s medium lacking the indicated amino acids for 30 minutes prior to lysis.

(c) Inhibition of translation with cycloheximide does not block inactivation of TORC1 upon amino acid withdrawal in Kc167 cells. Kc167 cells treated with either eIF4A dsRNA for 4 days, or cycloheximide (50 µg/mL, “CHX”) for 5 min prior to, as well as during removal of amino acids (-LIVASTQP). CHX treated cells still inactivate TORC1 (compare lanes 8 to 7) whereas eIF4A knockdown cells do not (lanes 14 vs 13). Quantifications of two biological replicates are shown. CHX and eIF4A samples were normalized to their respective control conditions.

(d) Knockdown of eIF4A in Kc167 cells does not prevent a drop in intracellular amino acids levels when amino acids are removed from the medium for 30 min. Quantification of total intracellular amino acids, analyzed in a blinded fashion. Levels of individual amino acids shown in Figure S2D. For CHX samples, cycloheximide (50µg/mL) was added 5 minutes prior to, and during treatment with medium containing or lacking amino acids. Statistical significance tested by ANOVA2 using Scheffé’s multiple comparisons method (p<0.05). Error bars: std. dev. n=5.
eIF4A inactivates TORC1 in response to amino acid starvation (2016)
Figure 3

Figure 3: The eIF4F complex regulates TORC1

(a) Knockdown of eIF4F components (eIF-4E and eIF-4G) partially phenocopy the eIF4A knockdown, leading to elevated TORC1 activity upon amino acid removal (30 min). Knockdown of all other tested translation initiation factors do not cause elevated TORC1 upon amino acid removal (main Figures 1c, 2a’ and 2b). Error bars: std. dev. n=3 biological replicates.

(b) Quantification of de novo protein synthesis rates by OPP incorporation reveals that eIF4E and eIF4G knockdowns deplete eIF4F function less efficiently than the eIF4A knockdown, explaining why the effects of eIF4E and eIF4G knockdowns on TORC1 activity (panel a) are a bit milder than the eIF4A knockdown. Kc167 cells treated with dsRNA for 4 days then incubated with 20μM Click-it OPP reagent 30min before fixation and fluorescent labeling. Quantification of OPP fluorescence per cell (nuclear count) for two independent experiments is displayed (3 independent images per condition), normalized to the no dsRNA condition. Scale bars: 25µm.
eIF4A inactivates TORC1 in response to amino acid starvation (2016)
Figure 4

Figure 4: TORC1 interacts physically with translation preinitiation complexes

(a) Binding of eIF4G to the Rag GTPases detected by co-immunoprecipitation from Kc167 cells in absence or presence of the chemical cross-linker DSP. Representative of >3 biological replicates.

(b) Binding between eIF4G and the Rag GTPase complex is mainly mediated by RagC, detected by expressing and immunoprecipitating either RagA or RagC alone. Representative of two biological replicates.

(c) Interaction between eIF4A and Raptor detected via co-immunoprecipitation of epitope-tagged proteins. Proteins were cross-linked with DSP prior to lysis and immunoprecipitation. Representative of 3 biological replicates.

(d-d’’) eIF4A and Raptor form a complex, detected by proximity ligation assay (PLA). Specificity of the PLA signal was controlled by knocking down either eIF4A or Raptor in Kc167. (d) Representative images. Cells outlined in white. (d’) Quantification of number of PLA spots per cell. (n>200. ***p<0.0001, ANOVA1, Dunnett’s multiple comparisons test, was performed on log-transformed data, box = +/- 1 quartile, whiskers = max/min). (d’’) Immunoblotting to detect efficiency of eIF4A and Raptor knockdowns.

(e-f) The eIF4A-Raptor interaction does not take place on lysosomes (e) or the endoplasmic reticulum (ER) (f). Kc167 cells were either incubated with 100 µg/mL Dextran for 1 hour, washed and incubated for 14 hours in normal growth medium to label late endosomes and lysosomes (e), or transfected to express an ER-resident GFP (f). Amino acid removal was for 45 min. Pearson's correlation coefficients for eIF4A-Raptor PLA and Dextran in panel (e) are 0.09 and 0.08 for +aa and -LIVASTQP respectively (calculated for all cells in the acquired fields, which were chosen randomly, n=40). Scale bars: 5µm.
eIF4A inactivates TORC1 in response to amino acid starvation (2016)
Figure 5

Figure 5: eIF4A regulates TORC1 via TSC2

(a) Knockdown of eIF4A and TSC2 do not elevate TORC1 activity in an additive manner, indicating they act in the same, and not parallel pathways. Whereas knockdown of eIF4A increases TORC1 activity in control Kc167 cells (lanes 1-2 and 5-6), eIF4A knockdown cannot further increase TORC1 activity in TSC2 knockdown cells (lanes 3-4 and 7-8). Error bars: std dev. n=3 biological replicates.

(b) Activation of TSC2 with the p90RSK inhibitor BI-D1870 rescues the elevated TORC1 activity caused by eIF4A knockdown in Kc167 cells, consistent with TSC2 acting downstream of eIF4A. BI-D1870 leads to inhibition of TORC1 (lanes 1-4) in a TSC2-dependent manner (see also Appendix Figure S5A). Representative of two biological replicates.

(c) FLAG-eIF4A and TSC2-V5 co-immunoprecipitate in Kc167 cells in the absence of chemical cross-linker. Cells were transfected with FLAG-eIF4A and TSC2-V5 expression vectors and treated with media containing or lacking amino acids for indicated timepoints prior to lysis and anti-FLAG immunoprecipitation. The experiment was performed in absence of the chemical cross-linker DSP. Representative of 3 biological replicates.

(d) Expression of TSC2-insensitive (S15H or Q63L), but not wild-type (WT) Rheb causes TORC1 activity to remain high upon amino acid removal. Kc167 cells were transfected to express either wild-type or mutant Rheb and then incubated with Schneider’s medium either containing or lacking amino acids for 30 minutes. Elevated TORC1 activity can be observed either by looking at phosphorylation of endogenous S6K, or phosphorylation of an HA-tagged S6K that was co-transfected with the Rheb constructs to assay specifically the transfected cells. Representative of two biological replicates.
eIF4A inactivates TORC1 in response to amino acid starvation (2016)
Figure 6

Figure 6: NAT1 regulates TORC1 activity via eIF4A

(a) Mass spectrometric analysis of proteins co-immunoprecipitating with eIF4A reveals NAT1 as one of the top interacting proteins. eIF4A immunoprecipitations were performed in triplicate, from cells treated with medium either containing (+aa) or lacking amino acids (-aa), and average values are shown. Peptide counts for each protein in each replicate were normalized to eIF4A peptide counts. Raw peptide counts shown in parentheses.

(a’) Schematic diagram of eIF4G and NAT1 primary protein structures. Binding sites for other initiation factors are shown.

(b) Binding between eIF4A and NAT1 is regulated by amino acid availability in a TORC1-independent fashion. Co-immunoprecipitation of tagged eIF4A and NAT1 in control Kc167 cells, or cells treated with medium lacking amino acids or supplemented with 20 nM rapamycin for indicated times. Representative of 3 biological replicates.

(c) Knockdown of NAT1 using 4 independent, non-overlapping dsRNAs leads to reduced TORC1 activity. Kc167 cells, treated with indicated dsRNAs for 4 days and then incubated with medium containing or lacking amino acids for 30 min. Error bars: std. dev. n=3 biological replicates.

(d) eIF4A is epistatic to NAT1 for TORC1 regulation. In the absence of amino acids, eIF4A knockdown cells have elevated TORC1 activity, NAT1 knockdown cells have reduced TORC1 activity, and eIF4A & NAT1 double-knockdown cells have elevated TORC1 activity compared to control cells. Cells were treated with indicated dsRNAs for 4 days and then treated with medium containing or lacking the indicated amino acids for 30 min. Error bars: std. dev. n=3 biological replicates.

(e) TSC2 is epistatic to NAT1 for TORC1 regulation. As in (d), knockdown of TSC2 rescues the reduced TORC1 activity caused by NAT1 knockdown.

(f) Schematic diagram showing the signaling relationships between NAT1, eIF4A and TSC2. When amino acids are present, NAT1 binds eIF4A and inhibits it. Upon amino acid removal, NAT1 releases eIF4A, which activates TSC2 to inhibit TORC1.
ATRX loss induces telomere dysfunction and necessitates induction of alternative lengthening of telomeres during human cell immortalization (2019)
Figure 1

Figure 1. Depletion of ATRX induces growth arrest and telomere dysfunction in human cells.

A Growth curves show proliferation reduction in ATRX depleted U87 cells. Data are expressed as means ± standard deviation (s.d.), N = 2. 500 cells/well were plated.

B β-gal staining revealed the increased senescent cell population in ATRX-deleted U87 cells. Upper panel, micrographs of control and ATRX-deleted U87 cells processed for ATRX immunofluorescence (IF) and β-gal senescence assay. Scale bar, 200 µm. D, DAPI. Lower panel, percentage of senescent cells. Data are expressed as means ± s.d., N = 3, unpaired t test.

C, D PML/TelG immuno-FISH (C) shows increased APB formation in ATRX-deleted U87 cells (D). Scale bar, 10 µm. (D) Percentages of APB positive cells are expressed as means ± s.d., N = 3, unpaired t test.

E C-circle assays revealed enhanced C-circles formation in ATRX-deleted U87 cells. Reactions lacking DNA or ɸ29 serve as negative controls. 30 ng of indicated template DNA was used in each reaction.

F, G 53BP1/TelG immuno-FISH (F) shows increased TIF formation in ATRX-deleted U87 cells (G). Arrows denote telomeres positive for 53BP1 signals. Scale bar, 10 µm. (G) Percentages of cells containing > 4 TIFs are expressed as means ± s.d., N = 3, unpaired t test.

H Growth curves revealed increase of cell proliferation in ATRX-deleted U87 cells (A-1 and A-2) after hTERT transduction. Data are expressed as means ± s.d., N = 2.

I Western blots show depletion of ATRX protein expression in hTERT-transduced and ATRX-deleted A-1 and A-2 U87 cells.
ATRX loss induces telomere dysfunction and necessitates induction of alternative lengthening of telomeres during human cell immortalization (2019)
Figure 2

Figure 2. Exogenous TERT expression mitigates ATRX depletion-induced telomeric dysfunction.

A Growth curves revealed that deletion of ATRX had minor effect on proliferation of TERT-overexpressed U87-T cells, but further removal of the exogenous floxed TERT by Ad-Cre induced rapid cell cycle arrest. Data are expressed as means ± s.d., N = 3.

B Telomere restriction fragment (TRF) analysis shows increased telomeric length heterogeneities in ATRX-deleted U87-T and the further enhancement following Ad-Cre treatment. Genomic DNAs (EtBr staining, left panel) prepared from control and ATRX-deleted U87-T cells at day 9 post control or Ad-Cre infection (middle panel) were assayed by hybridization with 32P-labelled (TTAGGG)4 probe, followed by re-hybridization with an oligonucleotide probe specific for centromere region (right panel). Genomic DNA from U2OS cells was used as an ALT positive control.

C, D PML/TelG immuno-FISH (C) shows increased APB formation in ATRX-deleted U87-T cells (D) at day 9 post control or Ad-Cre infection. Images are maximum-intensity projections of ~10 stacks captured with a 63x lens with 2x zoom. Scale bar, 10 µm. (D) Percentages of APB positive cells are expressed as means ± s.d., N = 3, unpaired t test.

E 2D gel analysis revealed increased T-circle formation in ATRX-deleted U87-T cells without or with Ad-Cre infection. Shown are gels stained with EtBr and blots hybridized with a TelG probe. Indicated are linear (lin) and open (oc) T-circle forms of telomeric DNA.

F C-circle assays shows increased C-circle formation in ATRX-deleted U87-T cells at day 9 post control or Ad-Cre infection.

G, H 53BP1/TelG immuno-FISH (G) shows increased TIF formation in ATRX-deleted U87-T cells at day 9 post control or Ad-Cre infection (H). Scale bar, 10 µm. (H) Percentages of cells containing > 4 TIFs are expressed as means ± s.d., N = 3, unpaired t test.
ATRX loss induces telomere dysfunction and necessitates induction of alternative lengthening of telomeres during human cell immortalization (2019)
Figure 3

Figure 3. Depletion of ATRX leads to progressive accumulation of TIFs and inhibition of cell growth.

A Schematic graph of structural domains of ATRX protein. Arrows indicated target sites of individual sgRNAs.

B Western blots of extracts from U87 cells infected with Lenti-CRISPR(puro) encoding individual ATRX-targeted sgRNAs. The extracts were isolated after puromycin selection, and assayed for ATRX depletion levels using antibody against ATRX or ACTIN control. The asterisks (*) denote the two sgATRXs (sgA#10 and sgA#19) used in following experiments.

C ATRX immunofluorescence staining of U87 cells infected with lentiviral control sgRosa, sgA#10, or sgA#19. D, DAPI. Scale bar, 50 µm.

D Schematic showing competition-based proliferation assay of individual sgRNAs. As sgRNA expression in Lentiviral LRG vector is linked to GFP, percentage changes of GFP+ cells reflect the fitness induced by CRISPR-based gene targeting.

E, F Bar graphs show time-course analyses of U87 (E) and U2OS cells (F) transduced with lentiviral LRG vector encoding sgRosa control or ATRX-targeted sgA#10 or sgA#19. The graph plots the percentage of GFP+ cells over time following transduction with indicated sgRNAs. Data are expressed as means ± s.d., * denotes ns (not significant); **, p < 0.01; ***, p < 0.001. N = 3, unpaired t test.

G, H 53BP1/TRF2 co-immunofluorescence (G) revealed strongly increased TIF formation in lentiviral sgA#10 or sgA#19 transduced U87 cells assayed at day 45 but not day 15 post infections (H). Scale bar, 15 µm. (H) Percentages of cells containing > 4 TIFs are expressed as means ± s.d., N = 3, unpaired t test.

I, J 53BP1/TelG immuno-FISH (I) revealed strongly enhanced TIF formation in lentiviral sgA#10 or sgA#19 transduced IMR90 cells assayed at day 35 but not day 10 post infections (J). Scale bar, 10 µm. (J) Percentages of cells containing > 4 TIFs are expressed as means ± s.d., N = 3, unpaired t test.

K Bar graphs show time-course analyses of IMR90 cells transduced with lentiviral sgRosa, sgA#10 or sgA#19. Data are expressed as means ± s.d., * denotes ns (not significant); **, p < 0.01; ***, p < 0.001. N = 3, unpaired t test.
ATRX loss induces telomere dysfunction and necessitates induction of alternative lengthening of telomeres during human cell immortalization (2019)
Figure 4

Figure 4. DAXX deletion induces ALT activation.

A, B γH2AX/TelG immuno-FISH (A) revealed increased TIF formation in IMR90 cells transduced with DAXX-targeted lentiviral sgRNAs (sgD#1 and #2) (B). Scale bar, 10 µm. Arrows denote telomeres that are positive for γH2AX signals. (B) Percentage of cells containing > 4 TIFs are expressed as means ± s.d., N = 3, unpaired t test.

C Western blots show depleted DAXX protein expression in Ad-Flp-infected cd-DAXX cells.

D, E PML/TelG immune-FISH (D) show enhanced APB formation in Ad-Flp infected cd-DAXX cells (cd-D#1 and cd-D#2) (E). The cells were analyzed at day 18 post Ad-Flp infection. Images are maximum-intensity projections of ~10 stacks captured with a 63x lens with 2x zoom. Scale bar, 10 µm. (E) Percentages of APB+ cells are expressed as means ± s.d., N = 3, unpaired t test.

F, G Depletion of DAXX induced delayed onset of growth arrest (F) and C-circle formation (G). (F) 2,000 cells were plated at day 4 post Ad-Flp infection. Data are expressed as means ± s.d., N = 3.