EMBO/soda-nel huggingface dataset:
Overexpression of Atg5 in mice activates autophagy and extends lifespan (2013)
f2

(a) Western blot analysis showing enhanced levels of Atg5, Atg12-Atg5 conjugate and LC3 conversion, and reduced p62 levels in the indicated tissues of 18-month-old Atg5 Tg mice no. 25 (T) and WT mice (W). (b) Densitometric analysis of the signals on the blots shown in (a). (c) Electron microscopy images of autophagosomes (arrows) and late-autophagic compartments (arrowheads) in the liver tissues of 18-month-old Atg5 Tg mice no. 25 and WT mice. Scale bars are indicated. (d) The numbers of autophagosomes and autolysosomes per cell (n=50) in the electron microscopic images shown in (c) were counted. The bars represent the mean±s.d. ***P0.0001 versus control; Student’s t-test. (e) Fluorogenic comparison of basal autophagy activity between GFP-LC3 Tg mice and Atg5/GFP-LC3 double Tg mice. Whole bodies of 8-week-old male (left) and female (right) GFP-LC3 Tg (LC3), and Atg5/GFP-LC3 Tg mice (Atg5/LC3) were visualized using a Kodak Image Station 4000MM equipped with a filter set for FITC (Kodak Molecular Imaging Software, Version 4.0). (f) Western blot analysis showing autophagy markers in the liver of GFP-LC3 mice. GFP-LC3 mice were injected for 5 days with siRNA-Atg5 or Atg5-HA cDNA through the tail vein. Whole-liver tissue lysates were prepared and subjected to immunoblotting using the indicated antibodies. β-Actin served as a control.
Overexpression of Atg5 in mice activates autophagy and extends lifespan (2013)
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(a) Representative physical pictures of 18-month-old WT and Atg5 Tg mice fed with a regular diet. (b) Body weight chart of ageing-induced obesity in WT and Atg5 Tg mice. The mice were fed with a regular diet for 24 months (n=180-–250) and their body weights were monitored monthly. The bars represent the mean±s.d. ***P0.0001 versus control; Student’s t-test. (c) Average daily oral food intake normalized to body weight, measured on regular diet. The food intake of mice (n=18–24) was measured for 2 weeks at 3-, 6-, 12- and 18-month-old age. (d) Quantification of gonadal fat pads from 18-month-old mice (n=11). ***P0.0001 versus control; Student’s t-test. (e,f) Quantification of visceral (e) and subcutaneous (f) fat pads of 18-month-old mice (n=6). ***P0.0001 versus control; Student’s t-test. (g) Enhanced glucose tolerance of Atg5 Tg mice. WT and Atg5 Tg mice were starved overnight and then given with an i.p injection of glucose (1 mg g−1 body weight). The results are the mean±s.d. of 12-week-old WT and Atg5 Tg mice (n=12). *P0.01, **P0.001, ***P0.0001 versus control; Student’s t-test. (h) Enhanced insulin sensitivity of Atg5 Tg mice. WT and Atg5 Tg mice were starved for 6 h and then i.p injected with 0.75 IU soluble insulin. *P0.01, **P0.001 versus control; Student’s t-test. (i) Plasma levels of triglycerol (TG) in WT and Atg5 Tg mice. The mice were fasted for 3 h before measurement. Young mice (4-week old, n=8); old mice (18-month old, n=8). ***P0.0001 versus control; Student’s t-test. (j) Plasma leptin levels in WT and Atg5 Tg mice (n=8–12). ***P0.0001 versus control; Student’s t-test. (k) GSH/GSSG ratio in the liver (n=4 males, three times trial). **P0.001, ***P0.0001 versus control; Student’s t-test. (l) Respiration rates of mitochondria in WT and Atg5 Tg MEFs (n=2). (m) Wire-hang endurance test of Atg5 Tg and WT mice (n=10). All the bars represent the mean±s.d. ***P0.0001 versus control; Student’s t-test.
Overexpression of Atg5 in mice activates autophagy and extends lifespan (2013)
f4

(a) Comparison of Atg12-Atg5 conjugate levels in WT and Atg5 Tg MEFs by western blot analysis. MEFs from WT and Atg5 Tg mice were cultured at embryonic day 13, after which cell extracts were examined by western blot analysis using anti-Atg5 and anti-β-actin antibodies. (b) Increased conversion of LC3 and reduced expression of p62 in Atg5 Tg MEFs no. 6 by rapamycin and/or Baf.A1. WT MEFs no. 1 and Atg5 Tg MEFs no. 6 in passage number 3 were incubated for 48 h with 10 μM rapamycin in the absence or presence of Baf.A1, and cell extracts were then analysed by western blotting. The signals on the blot were quantified by densitometric analysis and represented as the ratio of p62 to β-actin. (c,d) Increased resistance of Atg5 Tg MEFs to oxidative stress. WT MEFs no. 1 and Atg5 Tg MEFs no. 6 were treated for 24 h with 50, 100, 200 and 300 μM H2O2 (c) or 300 μM H2O2 in the absence or presence of 5 mM 3-MA or 20 nM Baf.A1 (Baf.A1) (d). Cell viability was assessed by propidium iodide staining. The values are the mean±s.e.m. (n=3). **P0.001, ***P0.0001 versus control; Student’s t-test. (e) Representative photographs showing the resistance of Atg5 Tg MEFs to oxidative stress. Primary cultured WT and Atg5 Tg MEFs were treated with 300 μM H2O2 for 24 h and then observed under a light microscope. Scale bars, 50 μm. (f) Effect of oxidative stress on the autophagy activity in Atg5 Tg MEFs. WT MEFs no. 1 and Atg5 Tg MEFs no. 6 were treated with 100 μM H2O2 for 24 h in the presence or absence of 20 nM Baf.A1, and cell extracts were analysed by western blotting. About 10 and 30 μg of proteins were used for LC3 (upper panel) and p62 (bottom panel) detection, respectively. β-Actin served as a control.
SMaRT lncRNA controls translation of a G-quadruplex containing mRNA antagonizing the DHX36 helicase (2020)
Figure 1

Figure 1 - Lnc-SMaRT functional characterization.

A Schematic representation of the lnc-SMaRT genomic locus. Genomic coordinates and exon/intron lengths are indicated. qPCR primers location is indicated by blue arrows while the regions corresponding to the siRNAs target sites are indicated by red lines.

B RT-PCR on cytoplasmic (C) and nuclear (N) extracts showing the subcellular localization of lnc-SMaRT. RNA was isolated from C2C12 cells after two days of differentiation. GAPDH mRNA and pre-mRNA (pre-GAPDH) were used respectively as cytoplasmic and nuclear controls. Representative results from three independent experiments are shown.

C qPCR analysis of lnc-SMaRT RNA expression in C2C12 cells undergoing differentiation at the indicated time points. The RNA expression levels in qPCR analyses were normalized against GAPDH mRNA and expressed as relative quantities with respect to GM samples set to a value of 1. Data are presented as the mean ± s.e.m. of three biological replicates (dots).

D qPCR on RNA extracts from C2C12 myoblasts treated with either control siRNAs (si-SCR) or with two different siRNAs against lnc-SMaRT (si-SMaRT-1, si-SMaRT-2) and induced to differentiate for two days. The RNA expression levels in qPCR analyses were normalized against GAPDH mRNA and expressed as relative quantities with respect to GM samples set to a value of 1. Data are presented as the mean ± s.e.m. of three biological replicates (dots). Statistical analysis was performed with ordinary analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. ***P < 0.001.

E Representative immunofluorescence for Myosin Heavy Chain (MHC in red) in combination with DAPI staining (in blue) C2C12 murine myoblasts transfected with either control (si-SCR) or lnc-SMaRT siRNAs (si-SMaRT-1, si-SMaRT-2) fixed after at two days of differentiation. Histograms represent MHC-positive mononucleated cells/total MHC-positive cells ratio and fusion index quantification (F.I.). At least 5 randomly chosen microscope fields of two independent biological samples were analyzed (n>600 cells for each field). Data are presented as the mean. of the two biological replicates (dots).
SMaRT lncRNA controls translation of a G-quadruplex containing mRNA antagonizing the DHX36 helicase (2020)
Figure 2

Figure 2 - lnc-SMaRT depletion impacts on C2C12 transcriptome.

A Gene expression heatmap showing the hierarchical clustering of protein-coding genes whose expression is altered upon lnc-SMaRT depletion. Expression values, calculated as RPKMs, were log2-transformed and mean centered. Only genes with an average RPKM between 10 and 1000 were plotted.

B Volcano plot describing the differential gene expression between si-SCR and si-SMaRT treated C2C12 samples. For each gene, log2 of fold-change versus -log10 of the unadjusted p-value is plotted. Points in red represent differentially expressed genes (FDR adjusted p-value < 0.05).

C Word cloud representing the Gene Ontology Biological Process terms enriched (Benjamini-Hochberg corrected p-value <0.05) in the lists of genes up-regulated (UP) or down-regulated (DOWN) upon lnc-SMaRT knockdown. The size of the words correlates with the Benjamini-Hochberg corrected p-value.

D Western blot on protein extract from control and lnc-SMaRT overexpressing C2C12 stable cell lines collected after one day of differentiation. CASP3 antibody was used. ACTN was used as loading control. Representative results from three independent experiments are shown.
SMaRT lncRNA controls translation of a G-quadruplex containing mRNA antagonizing the DHX36 helicase (2020)
Figure 4

Figure 4 - Representative images for MLX immunofluorescence on C2C12 myotubes.

A Immunofluorescence for total MLX protein detection (red signal) performed on C2C12 cells treated with siRNAs against Mlx-γ (si-Mlx-γ), lnc-SMaRT (si-SMaRT), DHX36 (si-DHX36) or with a control siRNA (si-SCR). Cells were fixed two days after the switch to differentiation medium. Representative binary images of single confocal planes for MLX immunofluorescence are shown for each treatment. The inserts on the topside of the images sketch the MLX signal peaks in the nuclei (white stars) to highlight the variation of MLX staining in each condition. Dashed lines indicate the edge of the nucleus.

B Histogram represents the fluorescence intensity ratio of the MLX protein signals in the nuclear (Nuc) and cytoplasmic (Cyt) compartment in the indicated conditions. About 50 cells from two independent experiments were analyzed as indicated in Materials and Methods. Data are presented as the mean of two biological replicates (dots).

C Venn diagram showing the overlap between genes deregulated upon lnc-SMaRT depletion and MLX targets [33], both in C2C12 samples. Genes at the intersection are those with opposite fold changes in the two depletion experiments. Only genes expressed in both systems were used to evaluate the overlap. Significance of overlap was evaluated using hypergeometric test.
SMaRT lncRNA controls translation of a G-quadruplex containing mRNA antagonizing the DHX36 helicase (2020)
Figure 5

Figure 5 - Lnc-SMaRT controls the translation of Mlx-γ through direct base pairing with the G4-containing region.

A Left panel: schematic representation of the luciferase constructs produced for Mlx. Mlx 5'- and 3'UTR-interacting regions were cloned respectively upstream and downstream the Renilla luciferase coding region (RLuc-Mlx 5'-3') and mutant derivatives devoid of the 5' interacting region (RLuc-Mlx-3') or the 3' UTR interacting region (RLuc-Mlx-5') were obtained. These constructs were co-transfected in C2C12 myoblasts in growth conditions together with plasmids expressing lnc-SMaRT (+) or with a control vector (-). Right panel: Luciferase activity data, presented as the mean ± s.e.m. of three biological replicates (dots), are shown with respect to each RLuc control vector (RLuc-Mlx 5'-3', RLuc-Mlx-3', RLuc-Mlx-5') set to a value of 1. Statistical analysis was performed with ordinary analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. *P < 0.05.

B The RLuc-Mlx-5' construct was co-transfected in N2a cells together with plasmids expressing lnc-SMaRT (+) or with a control vector (-) and treated with control siRNA (+) or siRNA against DHX36 (-). Luciferase activity data, presented as the mean ± s.e.m. of three biological replicates (dots), are shown with respect to RLuc-Mlx-5' vector set to a value of 1. Statistical analysis was performed with ordinary analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. **P < 0.01, ***P < 0.001.

C Left panel: schematic representation of the luciferase constructs used. The RLuc-Mlx-5' construct and its derived mutants (RLuc-Mlxmut30, RLuc-Mlx Δ75) were transfected in N2a cells in growth conditions together with lnc-SMaRT-expressing plasmids (lnc-SMaRT) or with its derived mutant (lnc-SMaRTmut) in the indicated combinations. Right panel: Luciferase activity data, presented as the mean ± s.e.m. of three biological replicates (dots), are shown with respect to each RLuc control vector (RLuc-Mlx-5', RLuc-Mlxmut30, RLuc-Mlx Δ75) set to a value of 1. Statistical analysis was performed with ordinary analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. ***P < 0.001.
SMaRT lncRNA controls translation of a G-quadruplex containing mRNA antagonizing the DHX36 helicase (2020)
Figure 6

Figure 6 - In vitro characterization of the interaction between lnc-SMaRT and Mlx-γ pairing sequences

A Upper panel: schematic representation of the predicted base pairing between lnc-SMaRT (region A) and Mlx γ mRNA; the predicted G-4 structure is boxed in red while the synthetic oligos sequences are underlined in red. Lower panel: Lane 1 and 2: 5 pmol of lnc-SMaRT and Mlx synthetic oligos were loaded on a denaturing polyacrylamide gel and stained for total RNA (SYBR GOLD, indicated as SYBR in the figure). Lane 3 and 4: 5 pmol of lnc-SMaRT and Mlx synthetic oligos were loaded on a native polyacrylamide gel and stained for total RNA (SYBR GOLD). Lane 5 and 6: 200 pmol of lnc-SMaRT and Mlx synthetic oligos were loaded on a native polyacrylamide gel and stained with the selective G-quadruplex staining N-methyl mesoporphyrin IX (NMM). Lane 7 and 8: 5 pmol of Mlx oligo alone (7) or in combination with 5pmol of lnc-SMaRT oligo (8) were heated at 100°C for 10 min and, after slow cooling, loaded on a native polyacrylamide gel and stained for total RNA (SYBR GOLD). Lane 9 and 10: 200 pmol of Mlx oligo alone (7) or in combination with 200 pmol of lnc-SMaRT oligo (8) were heated at 100°C for 10 min and, after slow cooling, loaded on a native polyacrylamide gel and stained with the selective G-quadruplex staining N-methyl mesoporphyrin IX. Asterisk (*) indicates the second NMM-positive band obtained upon SMaRT and γ-oligo interaction.

B 5 pmol of indicated oligos were loaded on a native polyacrylamide gel. Oligos were treated as described. °C = oligos were heated (+) or were not heated (-) at 100 °C for 10 min and loaded after slow cooling. KCl= oligos were incubated (+) or were not (-) in a buffer containing 100mM KCl (for G-quadruplex stabilization). The gel was stained with total RNA staining (SYBR GOLD).

C 200 pmol of indicated oligos treated as in (B) were loaded on a native polyacrylamide gel with the selective G-quadruplex staining N-methyl mesoporphyrin IX. Asterisk (*) indicates the second NMM-positive band obtained upon SMaRT and γ-oligo interaction.

Data information: Representative gels from at least three independent experiments are shown.

EXPANDED VIEW FIGURE LEGENDS
Apg10p, a novel protein‐conjugating enzyme essential for autophagy in yeast (1999)
Figure 1

Cloning of the APG10 gene and phenotype of Δapg10 cell. (A) Wild‐type (YW5‐1B), apg10‐1 mutant (MT91‐4‐2) and Δapg10 (TFD10‐L1) cells were incubated in SD(−N) medium containing 1 mM PMSF at 30°C. After incubation for 6 h, cells were observed under light microscopy (Nomarski images). (B) Wild‐type (closed circles), apg10‐1 mutant (closed triangles) and Δapg10 (open circles) cells were grown to 1 OD600/ml in YPD and then transferred to SD(−N) medium. After incubation at 30°C for the times indicated, their viability was determined by phloxine B staining (Tsukada and Ohsumi, 1993). (C) Cell lysates from wild‐type cells with empty vector pRS316 (lane 1) or HAAPG12 (lane 2), and also apg10‐1 (lane 3) and Δapg10 cells with HAAPG12 (lane 4) were subjected to Western blotting analysis with anti‐HA antibody (16B12). (D) Amino acid sequence of Apg10p. Cysteine residues are indicated in bold.
Apg10p, a novel protein‐conjugating enzyme essential for autophagy in yeast (1999)
Figure 2

In vivo interaction of Apg10p with Apg12p. (A) Two‐hybrid assay for interactions of Apg10p with Apg5p, Apg7p and Apg12p. Yeast cells (PJ69‐4A) harboring the indicated plasmids were streaked out on plates with medium lacking adenine to assay for interaction‐dependent activation of the ADE2 gene. (B) Total lysates from Δapg5 cells with HAAPG10 and/or MycAPG12 on 2μ plasmid were immunoprecipitated with anti‐HA (mAb 16B12) or anti‐Myc (mAb 9E10) antibody. Precipitates were analyzed by Western blotting using anti‐HA or anti‐Myc antibody. Apg12p adduct of Apg10p is indicated by ● and an unidentified complex of 67 kDa by ●●. The positions of cross‐reacting IgG heavy chain (H) and light chain (L) are indicated by arrowheads.
Apg10p, a novel protein‐conjugating enzyme essential for autophagy in yeast (1999)
Figure 3

Effects of the deletion of APG5 or APG7 on Apg12p–Apg10p thioester formation. Coimmunoprecipitation was performed with wild‐type (KA311B), Δapg5 (YNM122) or Δapg7 (YTS12) cells harboring HAAPG10 and MycAPG12 on 2μ plasmids. The resulting precipitates were analyzed by Western blotting. The DTT‐resistant Apg12p–Apg5p conjugate is indicated by ●.
Apg10p, a novel protein‐conjugating enzyme essential for autophagy in yeast (1999)
Figure 4

Apg12p is transferred to the Cys‐133 residue of Apg10p. (A) Cell lysates from Δapg10 cells (TFD10‐L1) harboring HAAPG12 and each mutant form of HAAPG10 on CEN plasmids were subjected to Western blotting analysis with anti‐HA antibody (mAb 16B12). (B) Cell extracts from the wild‐type cells (KA311B) harboring MycAPG12 and mutated HAAPG10 were subjected to immunoprecipitation and the resulting precipitates were analyzed by Western blotting with the rabbit anti‐HA antibody.
Apg10p, a novel protein‐conjugating enzyme essential for autophagy in yeast (1999)
Figure 5

The cysteine‐133 residue of Apg10p is essential for autophagy and the Cvt pathway. (A) Autophagic activities were measured by an ALP assay (Noda and Ohsumi, 1998). YTS3 cells (Δapg10 PHO8::pho8Δ60) harboring each mutant APG10 on CEN plasmids were grown to 1 OD600/ml in SC medium lacking tryptophan and then transferred to SD(−N) medium. Lysates from the cells after incubation for 0 and 4 h were used for assay. Error bars indicate the SD of three independent experiments. (B) Cell lysates from Δapg10 cells (TFD10‐L1) harboring empty vector, pRS316 (lane 1), wild‐type HAAPG10 (lane 2), HAAPG10C26S (lane 3), HAAPG10C133S (lane 4) or HAAPG10C137S (lane 5) on CEN plasmids were subjected to Western blotting analysis with anti‐API antiserum.
Munc13 activates the Munc18-1/syntaxin-1 complex and enables Munc18-1 to prime SNARE assembly (2020)
Figure 1

Figure 1 - Residues in the Munc18-1 domain 3a essential for MUN-catalyzed transition from the Munc18-1/Syx1 complex to the SNARE complex.

A Structure overview of the Munc18-1/Syx1 complex (pdb entry: 3C98). Mutating residues are highlighted in red below the domain depiction and shown as yellow sticks in the structure.

B Elution volumes (peak positions) of the Munc18-1 mutants in the absence (top) and presence (bottom) of Syx1 using size-exclusion chromatography on Superdex-200 column.

C Effects of the domain 3a mutations on MUN-catalyzed transition detected by native-PAGE assay. Components addition was indicated on the top of every lane.

D Quantitative analysis of the integrated densities of Munc18-1/Syx1 bands in C.

E Effects of the QKAA, KKEE and L348R mutations on MUN-catalyzed transition detected by FRET assay. Schematic diagram is shown on the top of the chart. FRET between BODIPY FL-labeled Syb2 (donor) and TMR-labeled SN25 (acceptor) was monitored.

F Effects of the QKAA, KKEE and L348R mutations on lipid mixing between liposomes with Munc18-1/Syx1 and liposomes containing Syb2 in the presence of Munc13 (M13), SN25 and Syt1/Ca2+. Illustration of the reactions is shown on the top of the chart.

Data information: Representative gels and/or traces displayed are from one of three independent replicates. In D−F, all bar charts are presented as mean values ± SD, n = 3 technical replicates.
Munc13 activates the Munc18-1/syntaxin-1 complex and enables Munc18-1 to prime SNARE assembly (2020)
Figure 2

Figure 2 - The Munc18-1 domain 3a is essential for synaptic vesicle exocytosis.

A Representative traces (left), summary graphs of normalized frequency (middle) and normalized amplitude (right) of mIPSCs recorded from cultured cortical neurons that were infected with lentivirus expressing Munc18-1 WT or mutants, with or without Munc18-1 shRNAs (Control, n = 25; None, n = 17; WT, n = 17; QKAA, n = 18; KKEE, n = 16; P335A, n = 23; L348R, n = 21).

B Sample traces (left), summary graphs of normalized amplitude (middle) and normalized charge transfer (right) of evoked IPSCs recorded from neurons as described in panel A (Control, n = 19; None, n = 19; WT, n = 19; QKAA, n = 13; KKEE, n = 18; P335A, n = 17; L348R, n = 21).

C Sample traces (left) and quantification of the normalized charge transfer evoked by hypertonic sucrose (right) recorded from the neurons described in panel A (Control, n = 17; None, n = 13; WT, n = 17; QKAA, n = 17; KKEE, n = 18; P335A, n = 15; L348R, n = 14).

Data information: In A−C, data are shown as mean values ± SEM; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Statistical significance was analyzed by Student's t-test. Number of neurons analyzed are from three independent cultures and data from different cultures are normalized. The raw data are shown in Appendix Fig S3.
Munc13 activates the Munc18-1/syntaxin-1 complex and enables Munc18-1 to prime SNARE assembly (2020)
Figure 3

Figure 3 - Monitoring Syx1 transport in HEK293T cell.

A Representative images of HEK293T cells transfected with mCherry2-Syx1 or EGFP-Munc18-1. Lipophilic dye DiD (1,1-Dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine) was used as the plasma membrane marker.

B Representative images of HEK293T cells co-transfected with mCherry2-Syx1 and EGFP labeled WT or mutants of Munc18-1.

C,D Summary graphs of the membrane distribution of Syx1 (C) and Munc18-1 WT or mutants (D) quantified by the ratio of fluorescence at the plasma membrane (PM) to the total fluorescence in the whole cell (total). DiD is the PM marker. Numbers of pictures analyzed are shown as follows: no Syx1, n = 14; no M18, n = 15; WT, n = 24; QKAA, n = 23; KKEE, n = 12; L348R, n = 22.

E Munc18-1 mutants retain interaction with Syx1 shown by GST pull-down experiment. A representative gel displayed is from one of three independent replicates.

Data information: In C and D, number of pictures analyzed are from three independent experiments; one picture contains one to three intact cells.. Data shown in summary graphs are mean values ± SEM. Statistical significance was analyzed by Student's t-test. ***, p < 0.001; ****, p < 0.0001. Scale bar: 20 µm. The scale bar applies to all images in each panel.