EMBO/soda-nel huggingface dataset:
FMRP recruitment of β-catenin to the translation pre-initiation complex (2018)
Figure 6

Figure 6: Wnt dependent cytoplasmic to nuclear translocation of β-catenin de-represses translation. A. (Top panel) Primary VSMCs were serum deprived overnight prior to stimulation with Wnt-3a (100 ng/mL) or its solvent (0.1% BSA in PBS) for 4 hr. The cells were treated with 0.5 µM puromycin for 15 min and harvested for Western blot analysis. Total β-catenin levels are indicated, and actin was used as a loading control. The average puromycin/actin ratio (n=3) was graphed. Error bars indicate standard deviation. An independent samples t-test was performed (2-tailed), with the p-value indicated above the error bar. (Bottom panel) Cells were treated similarly to above and fixed and stained for β-catenin (green) and nucleus (blue). B. The experiment in (A) was repeated using A10 cells. C. (i) HEK 293T cells were serum deprived for 1 hr and then stimulated with Wnt-3a (100 ng/mL) or its solvent (0.1% BSA in PBS) for 4 hr. The cells were harvested and subjected to an endogenous coIP analysis using either β-catenin or FMRP antibody. The corresponding proteins in the precipitated immunocomplex were detected by Western blot analysis. Non-programed rabbit IgG was used as a control. Efficacy of Wnt-3a treatment was assessed by inhibition of GSK3 mediated phosphorylation of β-catenin (S33/37/T41) by Wnt-3a. (ii) HEK 293T cells were serum deprived for 1 hr and then stimulated with Wnt-3a (100 ng/mL) or its solvent (0.1% BSA in PBS) for 4 hr. Cell lysates were then subjected to m7GTP-agarose pull-down analysis. eIF4E and tubulin were used as positive and negative controls, respectively, and phospho-β-catenin (S33/37/T41) Western blots were used as controls for Wnt-3a stimulation. D. A working model for the relief of β-catenin mediated translational repression by Wnt stimulation.

Legends for Expanded View Table and Figures

Table EV1: A list of 131 interacting partners identified by mass spectrometry and their corresponding peptide scores (as defined in Materials and Methods).
AGO2 promotes telomerase activity and interaction between the telomerase components TERT and TERC (2018)
Figure 1

Figure 1. AGO2 depletion in human cells reduces proliferation rate in vitro and tumor growth in vivo.

A. Proliferation curves of parental and AGO2KO HeLaS3 cells are plotted (n=6 experimental replicates).

B. Colony-forming activity of HeLaS3 and AGO2KO cells was determined by colony-formation assay (n=3 experimental replicates).

C. Tumour growth curves of xenografts derived from HeLaS3 or AGO2KO cells are shown as tumour diameter at different time points (n=5 mice for each group).

D. Xenograft tumour weights were measured 30 days post- injection (n=5 mice for each group).

Data information. Data are expressed as mean ± SEM. *P≤ 0.05; **P≤ 0.01; ***P≤ 0.01 (Student's t-test)
AGO2 promotes telomerase activity and interaction between the telomerase components TERT and TERC (2018)
Figure 2

Figure 2. AGO2 interacts with a sRNA arising from TERC locus (terc-sRNA)

A. TERC RNA secondary structure (adapted from http://telomerase.asu.edu). Terc- sRNA is highlighted in purple

B. HeLaS3 cells were transduced with a lentiviral vector coding for human TERC (HeLaS3_TERC) or for GFP, as a control (HeLaS3_GFP). The relative expression of TERC was measured by RT-qPCR. HPRT1 was used as internal control (n = 3 experimental replicates).

C. The relative expression of terc-sRNA was measured in HeLaS3_TERC and HeLaS3_GFP by RT-qPCR. RNU44 was used as internal control (n = 3 experimental replicates).

Data information. Data are expressed as mean ± SEM. *P≤ 0.05; ***P≤ 0.01 (Student's t-test).
AGO2 promotes telomerase activity and interaction between the telomerase components TERT and TERC (2018)
Figure 3

Figure 3. AGO2 controls telomere length and telomerase activity.

A. Average telomere length was measured from genomic DNA of parental and AGO2KO HeLaS3, by qPCR amplification of telomere repeats (T) and multicopy gene Alu (R), as a reference. The relative telomere length (T/R) was plotted (n=3 experimental replicates)

B. Representative images of metaphase spreads obtained from HeLaS3 and AGO2KO cells stained for telomeric sequences and centromere 2 alphoid DNA.

C. Representative telomere length distributions in parental and AGO2KO HeLaS3. Average telomere length and the standard deviation, as well as the total number of telomeres analyzed, are indicated. Notice the marked shift towards short telomeres in AGO2KO cells compared with the parental HeLa S3 cell line.

D, E. Telomerase activity was detected by TRAP in parental HeLaS3 and AGO2KO cells. 293T cells were used as a positive control. As negative controls, telomerase was heat- inactivated in cell extracts, in the last lane no cells were added in lysis buffer. Intensity of the telomerase products (6 bp-ladder) was determined by ImageLab Software (Biorad) and normalized with the intensity of the Internal Control (IC) (n=3 experimental replicates).

F. AGO2KO cells were transduced with a lentiviral vector coding for FLAG-HA-tagged AGO2 (AGO2KO_FH AGO2) or for GFP as a control (AGO2KO_GFP). Quantification of telomerase activity in AGO2KO_FH AGO2 and AGO2KO_GFP, as assessed by TRAP, was plotted (n=3 experimental replicates).

G. HeLaS3 cells were transduced with a lentiviral vector coding for FLAG-HA-tagged AGO2 (HeLaS3_FH AGO2) or for GFP as a control (HeLaS3_GFP). Quantification of telomerase activity in HeLaS3_FH AGO2 and HeLaS3_GFP, as assessed by TRAP, was plotted (n=3 experimental replicates).

Data information. Data are expressed as mean ± SEM. *P≤ 0.05; **P≤ 0.01; ***P≤ 0.01 (Student's t-test)
AGO2 promotes telomerase activity and interaction between the telomerase components TERT and TERC (2018)
Figure 4

Figure 4. AGO2 expression does not affect neither TERC RNA levels and 3'-end processing nor TERT protein abundance, but controls association between TERT and TERC.

A. The relative expression of TERC was measured by RT-qPCR from total RNA of HeLaS3 and AGO2KO cells. HPRT1 was used as internal control (n = 3 experimental replicates).

B. qPCR of 3'-extended TERC transcript in cDNA from HeLaS3 and AGO2KO cells generated using random hexamers. HPRT1 was used for normalization (n=3 experimental replicates).

C. qPCR of TERC transcript in cDNA from HeLaS3 and AGO2KO cells generated using oligo(dT)10 priming. HPRT1 was used for normalization (n=3 experimental replicates).

D. Whole cell extract of HeLaS3 and AGO2KO was analyzed by western blot for the presence of TERT protein. GAPDH was used as loading control. Data are representative of 4 independent experiments.

E. TERT protein level was quantified by ImageLab Software (Biorad). GAPDH was use as internal control (n=4 experimental replicates)

F-G. HeLaS3 and AGO2KO cell extracts (n=3), as well as AGO2KO_FH AGO2 and AGO2KO_GFP cell extracts (n=3 experimental replicates) were immunoprecipitated using an anti-TERT antibody or IgG as mock IP. TERC and HOTAIR (as a negative control) abundance was assessed by RT-qPCR. HPRT1 was used for normalization and enrichment in TERT-RIP as compared to IgG RIP was plotted.

H. HeLaS3 whole-cell extract was immunoprecipitated using anti- AGO2 antibody or IgG, as mock IP. Whole cell lysates (input) and immunoprecipitates were analyzed by western blot with anti-TERT antibody. As a positive control, the presence in immunoprecipitates of GW182, a known AGO2-interacting protein, was assessed. Data are representative of three independent experiments.

Data information. Data are expressed as mean ± SEM. *P≤ 0.05; ns= not significant (Student's t-test)
AGO2 promotes telomerase activity and interaction between the telomerase components TERT and TERC (2018)
Figure 5

Figure 5. AGO2 is bound to TERC RNA and deletion of TERC regions complementary to terc-sRNA impairs AGO2/TERC interaction.

A. HeLaS3 whole-cell extract was immunoprecipitated using anti- AGO2 antibody or IgG, as mock IP. TERC RNA enrichment in AGO2 RIP as compared to IgG RIP was assessed by RT-qPCR . 7SK RNA was used for normalization (n=4 experimental replicates).

B. RIP assay was performed from HeLaS3_FH AGO2 and HeLaS3_GFP cell extract using anti-HA antibody or IgG, as negative control, followed by TERC detection by RT-qPCR. 7SK RNA was used for normalization (n=3 experimental replicates).

C. HeLaS3 cells were transduced with a lentiviral vector coding for wild-type TERC (HeLaS3_TERCwt), TERC mutated in position 313-340 (HeLaS3_TERC (313-340mut)) or TERC delated in position 12-31 (HeLaS3_TERC (Δ 12-31)). Whole-cell lysates from HeLaS3_TERCwt, HeLaS3_TERC(313-340mut) and HeLaS3_TERC (Δ 12-31) was immunoprecipitated using an anti-AGO2 antibody in order to assessed the impact of TERC mutations on AGO2 binding. For each experiment the enrichment of ectoTERC as compared to 7SK RNA in RIP samples was normalized on the amount of ectoTERC and 7SK RNA in input samples. Association of AGO2 with TERC(313-340mut and TERC (Δ 12-31) was compared to the association with TERCwt (n=3 experimental replicates).

Data information. Data are expressed as mean ± SEM. *P≤ 0.05; **P≤ 0.01; ***P≤ 0.01 (Student's t-test)
AGO2 promotes telomerase activity and interaction between the telomerase components TERT and TERC (2018)
Figure 6

Figure 6. Terc-sRNA overexpression increases telomerase activity even in the absence of AGO2.

A-B. HeLaS3 and AGO2KO cells were transfected with a synthetic terc-sRNA or an siRNA with no target in humans (siCtl), as a control. Four days after transfection, telomerase activity in 100- and 50 cell-lysate was assessed by TRAP. Intensity of the telomerase products (6 bp-ladder) was determined by ImageLab Software (Biorad) and normalized with the intensity of the Internal Control (IC), (n=4 experimental replicates)

Data information. Data are expressed as mean ± SEM. *P≤ 0.05; **P≤ 0.01 (Student's t-test)

Expanded View Figure Legend
Lysosomal dysfunction disrupts presynaptic maintenance and restoration of presynaptic function prevents neurodegeneration in lysosomal storage diseases (2016)
Figure 2

Figure 2. Presynaptic alterations are recapitulated in cultured MPS-IIIA neurons and are induced by lysosomal inhibition in healthy neurons.

(A) Synaptic terminal endocytosis was analyzed in WT and MPS-IIIA DIV14 hippocampal neurons by quantification of incorporated FM1-43 dye fluorescence in ~300 individual boutons (taken from 4-5 coverslips for each group). Fluorescence intensities were expressed as arbitrary units (A.F.U.) and displayed both as a distribution and as mean values ± s.e.m.

(B) WT and MPS-IIIA hippocampal neurons (DIV14) were transfected with v-Glut1-pHluorin-mCherry plasmid and synaptic recycling was evaluated by the fluorescence change of the probe from 80-100 individual boutons (taken from 4-5 coverslips for each group). Fluorescence intensity was quantified at each bouton during KCl perfusion, normalized to the fluorescence value obtained upon rapid alkalization (NH4Cl perfusion) and expressed as ∆fluorescence (normalized to baseline; ΔFKCl/ΔF NH4Cl). Values were displayed both as fluorescence traces (means ± s.e.m. at each time frame) and maximum fluorescence after KCl perfusion (means ± s.e.m.). Representative panels on the right show the fluorescence intensity change in control neurons (WT). Pseudo-colour was applied to better reveal fluorescence changes.

Note that NH4Cl alkalinizes all vesicles revealing the total (recycling + resting) pool in neuronal cells analyzed.

(C) WT hippocampal neurons at DIV10 were treated with a lysosome inhibitor’s cocktail for 3 days. The size of lysosomal compartment was evaluated in both treated and control untreated WT neurons by EM examination. Quantitation analysis was performed on 30 different images.

(D, E) Endocytosis (D) and exocytosis (E) in presynaptic boutons from treated and control untreated WT hippocampal neurons were monitored as in (A, B).

(F) EM analysis of synaptic terminals was performed in treated and control untreated WT neurons. The number of synaptic vesicles per synapse was quantified from ~20 different images (taken from 3 ultrathin sections for each group), normalized by the length of synaptic cleft and expressed as % of WT. Arrows indicate the synaptic cleft.

Data are means ± s.e.m. *P<0.05, Student’s t-test: MPS-IIIA vs WT (A, B). WT + lys. block (72h) vs control WT (C-F). Scale bar 4µm (A, D); 0.2 µm (C); 0.1 µm (F).
Lysosomal dysfunction disrupts presynaptic maintenance and restoration of presynaptic function prevents neurodegeneration in lysosomal storage diseases (2016)
Figure 3

Figure 3. Proteostasis of the machinery components mediating the synaptic vesicle recycling is altered in MPS-IIIA mice

(A) VAMP2, SNAP-25 and Syntaxin-1 SNAREs were immunoblotted in WT and MPS-IIIA synaptosomal brain samples at indicated ages. Synapsin-1 was also blotted as a control. Quantitation of WB was shown.

(B) Confocal microscopy images of WT and MPS-IIIA hippocampal neurons (DIV14) double labeled with anti-synapsin-1 (presynaptic marker; red) and either anti-VAMP2 or anti-SNAP25 antibodies (green). The merges (yellow) of confocal images are shown. SNAP25-synapsin-1 and VAMP2–synapsin-1 co-localizations were quantified using the Manders' Colocalization Coefficients (MCC) (ImageJ) and displayed as % (MCC x 100) of Synapsin-1 co-localizing with either SNAP25 or VAMP2 (means ± s.e.m. from 15 different images taken from 4-5 coverslips for each group).

(C) VAMP2 and SNAP-25 protein levels were quantified by immunoblot analysis in WT and MPS-IIIA hippocampal neurons (DIV14) at different times after cycloexamide treatment and expressed as % of remaining protein at T0 (100 %). The proteasome was inhibited as indicated.

(D) SDS-resistant complex levels were evaluated in WT and MPS-IIIA total brain samples at indicated ages by immunoblotting of non-boiled samples with VAMP2 or Syntaxin-1 antibodies. Quantitation of WB was shown.

(E) Total brain lysates were immunoprecipitated with antibodies to Syntaxin-1 and co-immunoprecipitated VAMP2 and SNAP25 proteins were revealed by WB analysis. The levels of immunoprecipitated proteins were quantified.

(F) α-Synuclein and CSPα were immunoblotted in WT and MPS-IIIA total brain lysates at indicated ages Total protein levels were quantified.

(G) α-Synuclein and CSPα were immunoblotted in WT and MPS-IIIA synaptosomal fractions at indicated ages. Hsc70, an heat shock cognate protein, which forms together with CSPα and SGT (small glutamine-rich tetratricopeptide repeat domain protein) the “chaperon machine” was also blotted. Protein levels were quantified.

Data are means ± s.e.m. N=3 (biological triplicates) in WB quantitation. *P<0.05, Student’s t-test: MPS-IIIA at each age vs WT (A, D, F, G); MPS-IIIA vs WT (B, E); Either WT or MPS-IIIA + prot. inhib. (at each time-point) vs MPS-IIIA (C). Scale bar: 5 µm (B).
Lysosomal dysfunction disrupts presynaptic maintenance and restoration of presynaptic function prevents neurodegeneration in lysosomal storage diseases (2016)
Figure 4

Figure 4. Lysosomal-driven deregulation of α-synuclein and CSPα degradation

(A) WB analysis of LC3 and p62 (an autophagy substrate) was performed on WT and MPS-IIIA brain samples at indicated ages. WB quantitation was shown.

(B) α-Synuclein was immunoblotted in WT and MPS-IIIA both total and synaptosomal brain fractions at indicated ages after sequential extraction with detergents with increased strength. Soluble (Sol.), lowly insoluble (L. Insol.) and highly insoluble (H. Insol.) forms correspond to the protein solubilized respectively in Triton-X100, SDS 10% and 8M UREA.

(C) Co-immunofluorescence analysis of α-synuclein with SMI-32 in WT and MPS-IIIA hippocampal neurons (DIV14). α-Synuclein synaptic puncta present in a neurite tract of 10µm is showed in a representative enlarged image. Quantification of α-synuclein synaptic puncta was calculated from 30 different enlarged images c.

(D) Confocal analysis of α-synuclein (green) and LAMP1 (red) in WT and MPS-IIIA hippocampal neurons (DIV14). Enlarged merge images are also shown. Co-localization was quantified using the MCC coefficient (ImageJ) and displayed as % (MCC x 100) of α-synuclein co-localizing with LAMP1 (means ± s.e.m. from 15 different images taken from 4-5 coverslips for each group).

(E) CSPα was immunoblotted in WT and MPS-IIIA total brain lysates at indicated ages after sequential extraction with detergents with increased strength as in (B).

(F) Co-immunofluorescence analysis of CSPα and SMI-32 in DIV14 hippocampal neurons. CSPα- synaptic puncta were quantified as in (C).

(G) CSPα protein levels were evaluated by immunoblot analysis in WT and MPS-IIIA hippocampal neurons (DIV14) at different times after cycloexamide treatment and expressed as % of remaining protein at T0 (100 %). The proteasome was inhibited as indicated.

(H) Palmitoylation-dependent shift in the molecular weight of CSPα was evaluated in WT and MPS-IIIA brain samples at indicated ages by immunoblotting CSPα in boiled samples prepared without exposure to sulphydryl agents (beta-mercaptoethanol or dithiothreitol).

(I) The protein levels of RPN10 (the regulatory subunit of 26S proteasome) and ubiquitinated proteins formed by Lys-48 (K48) residue linkage (involved in protein degradation via the proteasome) were evaluated in WT and MPS-IIIA brain samples at indicated ages by WB analysis. WB quantitation was shown.

(L) Proteasome activity was evaluated by measuring the chymotrypsin-like activity in WT and MPS-IIIA mouse brain samples at different ages. Proteasome activity was expressed as % of WT activity

Data are means ± s.e.m. N=3 (biological triplicates) in WB quantitation. *P<0.05, **P<0.001 Student’s t-test: MPS-IIIA at each age vs WT (A, I, L); MPS-IIIA vs WT (C, D, F); Either WT or MPS-IIIA + prot. inhib. (at each time-point) vs MPS-IIIA (G). Scale bar: 10 µm (C, D, F).
Lysosomal dysfunction disrupts presynaptic maintenance and restoration of presynaptic function prevents neurodegeneration in lysosomal storage diseases (2016)
Figure 5

Figure 5. α-Synuclein- and CSPα-dependent defective proteostasis of SNAREs is recapitulated in healthy neurons upon lysosomal inhibition.

(A) Autophagy was monitored in WT hippocampal neurons treated with the lysosome inhibitor’s cocktail for 3 days by immunoblot analysis with anti-LC3 antibodies. As control DIV10 WT neurons were left untreated and monitored by LC3 blot over the three days period. The LC3-II levels quantitation was shown.

(B) Proteasome activity was measured in treated and control untreated WT neurons using pZsProSensor-1 vector (green). Cells were co-stained with anti SMI-32 (blu). Proteasome activity was quantified by measuring the green fluorescence (inversely correlated with proteasome activity) in 10 different cells (taken from 4-5 coverslips for each group) and expressed as fold to WT.

(C) Activation of calpain 2 and caspase-3 proteolytic systems was evaluated by WB measurement of the protein levels of calpain 2 and caspase-3 (both full-length and activated cleaved forms of ~17 KDa) in treated and control untreated WT neurons. Arrow indicated the full-length caspase-3 protein.

(D) After 3 days-treatment cell lysates from treated and control untreated WT neurons were immunoblotted with α-synuclein and CSPα and protein levels were quantified.

(E) Treated and control untreated WT neurons were subjected to α-synuclein (green) and LAMP1 (red) confocal analysis. Enlarged merge images are also shown. Co-localization was quantified using the MCC coefficient (ImageJ) and displayed as % (MCC x 100) of α-synuclein co-localizing with LAMP1 (means ± s.e.m. from 15 different images taken from 4-5 coverslips for each group). Cells were also co-stained with anti SMI-32.

(F) Treated and control untreated WT neurons were subjected to CSPα/SMI-32 co-immunofluorescence. CSP synaptic puncta present in a neurite tract of 10µm is showed in a representative enlarged image. Quantification of CSPα synaptic puncta was calculated from 30 different enlarged images taken from 4-5 coverslips for each group.

(G) SDS-resistant complex levels in treated and control untreated WT neurons were evaluated by immunoblotting analysis of non-boiled samples with VAMP2 or SNAP-25 antibodies. The amounts of SNARE complexes were quantified.

Data are means ± s.e.m. N=3 (biological triplicates) in WB quantitation. *P<0.05, **P<0.001 Student’s t-test: WT + lys. block (at each time-point) vs control WT (A). WT + lys. block (72h) vs control WT (B-G). Scale bar: 5 µm (B); 10 µm (E, F).
Lysosomal dysfunction disrupts presynaptic maintenance and restoration of presynaptic function prevents neurodegeneration in lysosomal storage diseases (2016)
Figure 6

Figure 6. CSPα and α-synuclein synergically contribute to defective SNARE proteostasis in genetically modified mice

(A) α-Synuclein, CSPα, VAMP2 and SNAP-25 were immunoblotted in total brain homogenates and/or synaptosomal fractions of 1.5 month-old WT (CSPα+/+/α-syn+/+), CSPα-/-, CSPα+/-, α-syn-/-, α-syn+/- and CSPα+/-/α-syn+/-. Synapsin-1 was also blotted as a control. Quantitation of WB in synaptosomal samples was shown.

(B) SDS-resistant complex levels were evaluated in brain homogenates derived from mice with indicated genotypes by immunoblotting of non-boiled samples with VAMP2 or Syntaxin-1 antibodies. Quantitation of WB was shown.

(C) EM analysis of cortical synapses derived from mice with indicated genotypes. The number of synaptic vesicles per synapse was quantified from 40 different images (taken from 5 different mice for each genotype), normalized by the length of synaptic cleft and expressed as % of WT. The size of synaptic vesicles was quantified from 400-500 vesicles (taken from 5 different mice for each genotype) and expressed as the average of vesicle diameter (nm) Arrows indicate the synaptic cleft.

(D) Extracellular recordings (fEPSPs) in hippocampal brain slices from 1.5 months-old WT (CSPα+/+/α-syn+/+) and CSPα+/-/α-syn+/- mice. The left panel shows representative fEPSP traces. Summary graphs show the fEPSPs slope as a function of either the applied stimulus intensity or the prevolley amplitude. Data are the average of the values from 5 slices (from 4 mice) in the WT group and 5 slices (from 4 mice) in the CSPα+/-/α-syn+/-. *p<0.05, repeated measure ANOVA.

Data are means ± s.e.m. N=3 (biological replicates) in WB quantitation (A, B). *P<0.05, **P<0.001 Student’s t-test: each genotype vs WT (A, B). Scale bar: 0.2 µm (C)