EMBO/soda-nel huggingface dataset:
TFG clusters COPII‐coated transport carriers and promotes early secretory pathway organization (2014)
Figure 7

TFG depletion causes the accumulation of cargo‐laden COPII transport carriers throughout cells

Human RPE‐1 cells stably expressing low levels of mannosidase II‐mApple were transfected with a TFG siRNA for 60 h. Cells were subsequently treated with brefeldin A (BFA) for 1 h, followed by a wash into fresh media and further incubation for 2 h in the absence of BFA, prior to fixation. Images shown are projections of 3D data sets (4 μm in z). Merged images with Sec31A in green and ManII in red are shown (representative of at least 15 cells analyzed). Scale bar, 5 μm.
Human RPE‐1 cells stably expressing low levels of mannosidase II‐mApple were transfected with a TFG siRNA for 60 h. Cells were subsequently treated with BFA for 1 h, followed by a wash into fresh media and further incubation for 1 h in the absence of BFA, prior to fixation and staining using α‐Sec16A and α‐Sec31A antibodies. Images shown are projections of 3D data sets (4 μm in z). Merged images with Sec31A (green), ManII (red), and Sec16A (blue) are shown (representative of at least 15 cells analyzed). Scale bar, 5 μm.
Higher magnification views of the indicated regions in (B, boxed) are shown. Arrows highlight COPII‐positive transport carriers that contain the cargo ManII, which are not juxtaposed to Sec16A‐labeled sites on the ER. Additionally, arrowheads point out distinct foci in which COPII continues to associate with Sec16A‐labeled sites, indicating that COPII vesicle formation continues in the absence of TFG. Scale bar, 1 μm.
Prevention of dsRNA-induced interferon signaling by AGO1x is linked to breast cancer cell proliferation (2020)
Figure 1

Fig 1 Evidence of AGO1 transcript translational readthrough and of AGO1x expression.

A, Top: schema of analyzed TR regions (purple), located downstream of the annotated open reading frame (grey), between the annotated stop codon (red triangle) and the next in-frame stop codon (orange triangle); Bottom: histogram of average PhastCons conservation scores (x-axis) of putative TR regions of all RefSeq-annotated transcripts. The scores of the four human Argonaute protein family members are highlighted.

B, Multiple sequence alignment of the AGO1 putative TR region across vertebrates.

C, Multiple sequence alignment of the corresponding predicted amino acid sequence. The unique peptide targeted by the polyclonal antibody is indicated by the red line. The green and blue lines indicate peptide sequences obtained after tryptic digestion, in which cleavage is exclusively at Arginine (R) and Lysine (K) (further described below in panels E and F). Red asterisks indicate stop codons.

D, Western blot showing AGO1x expression in three cell lines. For comparison, a parallel blot was probed with an antibody directed against canonical AGO1. Tubulin served as loading control.

E,F, Annotated MS/MS spectrum of peptides specific for the endogenous AGO1x, "QNAVTSLDR", depicted in green (E) and "LSKPQELCHPNPEEAR", depicted in blue (F). The Mascot Ion Score (text color corresponds to peptides marked in Fig 1C for reference) as well as the annotated fragments (blue = y-ions; red = b-ions) together with the corresponding amino acids are indicated.
Prevention of dsRNA-induced interferon signaling by AGO1x is linked to breast cancer cell proliferation (2020)
Figure 2

Fig 2 AGO1x localizes to the nuclear region

A, Representative immunofluorescence images showing the subcellular distribution of AGO1x (red) relative to nuclear and cytoplasmic markers. DAPI was used to mark the nucleus (blue). The co-stained subcellular marker is indicated in each panel in green. SC 35, Lsm4, α-Tubulin, ERP72, p54 (NRB), and Nucleolin serve as markers for nuclear speckles, cytosol and nucleus, cytosol, endoplasmic reticulum, paraspeckle and nucleolus, respectively.

B, Mean (+/- s.d.) pixel intensities of AGO1x staining in nucleolus and nucleoplasm, computed from z-stack images of MDA-MB-231 cells (n=20). The P-value was determined using a paired two-tailed t-test.

C, Representative AGO1x and AGO1 blot from MDA-MB-231 cell fractions. GAPDH and hnRNP C1/C2 served as markers of purity of the individual fractions.
Prevention of dsRNA-induced interferon signaling by AGO1x is linked to breast cancer cell proliferation (2020)
Figure 3

Fig 3 AGO1x promotes cell proliferation.

A, Top: Scheme of the sites targeted by sgRNA in the generation of AGO1x mutant cell lines. Bottom: Western analysis of lysates from mutant MDA-MB-231 cells showing the depletion of AGO1x and the comparable total AGO1 levels across the cell lines. Three independently collected sets of cells were lysed and analysed in parallel. After calculating the levels of AGO1x and AGO1 relative to GAPDH, values corresponding to individual lanes were normalized to the average over the replicates of the control line. These numbers are indicated below the blots. The P-values of the t-tests comparing the expression AGO1x in mutant (W1A and W6A) and control (CTRL) lines were W1A-CTRL: 0.0008, W6A-CTRL: 0.0002, and for AGO1 W1A-CTRL: 0.1227, W6A-CTRL: 0.1173.

B, Phase contrast images of control and mutant cell lines at 24 and 48 hr after plating equal numbers (10'000) of cells in individual wells of a six well plate.

C, Impedance-based mean (+/- s.d.) cell indices at the indicated time points after seeding equal numbers of CTRL (n=6), W1A (n=5) and W6A (n=5) cells. From 6 hours on, there is a statistically significant difference between control and the two mutant cell lines (P < 0.001, two tailed t-test).

D, Impedance-based mean (+/- s.d.) cell indices as a function of time for control (n=3), W1A (n=4) and W6A (n=3) cells grown in electronically monitored Boyden chambers. From 12 hours on, there is a statistical significant difference between control and the two mutants (P < 0.005, two tailed t-test).

E, Mean (+/- s.d.) number of colonies obtained in a soft agar colony formation assay of control, W1A and W6A mutant cell lines (all n=4, P-value determined using an unpaired two-tailed t-test).

F, Mean (+/- s.d.) number of spheres obtained from a sphere formation assay of control, W1A and W6A mutant cell lines (all n=8, P-values computed as in E).

G, AGO1x staining of tissue sections from breast cancers, classified as low-proliferating or high-proliferating based on Ki-67 staining. The upper and lower panel represents two different tissue sections.

H, Mean (+/- s.d.) percentage of AGO1x positive cells in breast tumors with low (n=7) and high (n=9) proliferation index, assessed by the expression level of Ki-67. P-value was determined using an unpaired two-tailed t-test.
Prevention of dsRNA-induced interferon signaling by AGO1x is linked to breast cancer cell proliferation (2020)
Figure 4

Fig 4 Depletion of AGO1x leads to increased interferon response and apoptosis.

A, Scatter plot of mean log2 mRNA fold-changes in the mutant cell lines versus control (n=3). Shown are also the Pearson correlation coefficient and respective P-value. The dashed line indicates equal change in the two mutant lines.

B, Normalized enrichment score (ES) from gene set enrichment analysis (Subramanian et al, 2005) comparing gene expression changes in the mutant cell lines relative to the control cell line. For all depicted pathways, P < 0.05. P-values were calculated by comparing the empirical ES of a gene set to a null distribution of ESs derived from permuting the gene set, and then adjusted for multiple hypothesis testing.

C, Mean (+/- s.d.) activity of STAT1/IRF1/IRF2/IRF3/IRF9 transcription factor motifs estimated by ISMARA (Balwierz et al, 2014) (n=3). Shown are the P-values (*** P < 0.001) determined by Dunn's multiple comparisons test post hoc and the non-parametric Kruskal-Wallis test (P < 0.0001).

D, Mean (+/- s.e.m.) log2 mRNA expression fold-changes (inferred from RNA-Seq) of genes involved in the interferon alpha response and apoptosis in the two mutant cell lines relative to control (n=3). Multiple-testing-corrected P-values for Wald tests comparing fold-changes with respect to control are depicted above each bar (** P < 0.01, *** P < 0.001).

E, Mean (+/- s.d.) expression fold-changes of the corresponding proteins (if detected in the proteomics data) in the two mutant cell lines (W1A, n=2; W6A, n=1) compared to Control (n=2). Multiple-testing-corrected P-values for unpaired two-tailed t-tests (proteomics) comparing fold-changes with respect to control are depicted above each bar (* P < 0.05, ** P < 0.01, *** P < 0.001).

F, Representative result of the apoptosis assay using AnnexinV and propidium iodide (PI) staining in the control and the two mutant cell lines (left). The percentage of cells in each quadrant is depicted for each cell line. Quantification of the mean (+/- s.d.) percentage of apoptotic cells (Q1+Q2, AnnexinV+) across the different cell lines (n=4) (right). Shown is the P-value determined by unpaired two-tailed t-test.

G, Impedance-based mean (+/- s.d.) cell index values at the indicated time points of growth after seeding equal numbers of control, W1A and W6A cells, upon treatment with DMSO or Ruxolitinib. For the DMSO treatment: Control (n=4), W1A (n=3) and W6A (n=3). For the Ruxolitinib treatment: Control (n=6), W1A (n=3) and W6A (n=4). From 6 hours after DMSO treatment, there is a statistically significant difference between control and the two mutants (P < 0.05, two tailed t-test), whereas no statistically significant difference is found after Ruxolitinib treatment.
Prevention of dsRNA-induced interferon signaling by AGO1x is linked to breast cancer cell proliferation (2020)
Figure 6

Fig 6 AGO1x interacts with PNPT1 to buffer endogenous dsRNA levels and regulate growth

A, Summary of mass-spectrometric analysis of AGO1x (n=5) or IgG (n=2) IP from control cells. P-values were calculated using Fisher's exact test and corrected for multiple testing using the Benjamini-Hochberg method.

B, Western blot analysis to validate the interaction of AGO1x with PNPT1 and DHX9 in control MDA-MB-231cells. AGO1x pulldowns were compared to IgG pulldowns to verify the relative levels of PNPT1 and DHX9 from four independent biological replicates. The immunoglobulin heavy chain band served as a loading control.

C,D, Validation of AGO1x-PNPT1 interaction by reciprocal pulldown. Immunoprecipitation of PNPT1 from control and mutant MDA-MB-231 cells and western blotting to validate the specificity of PNPT1 antibody in comparison to IgG control (C). Presence of AGO1x and Fibrillarin in PNPT1 immunoprecipitates. Three independent biological replicates were used, and the eluted proteins were analyzed by western blotting (D).

E, in vitro interaction assay of isolated nuclear extracts from control MDA-MB-231 cells with FLAG-tagged AGO1 or FLAG-tagged AGO1x immunoprecipitated from cells stably expressing the constructs. Table indicates the peptide counts from a second round of immunoprecipitation with anti-FLAG antibody and subsequent analysis by LC-MS/MS. P-values are from Fisher's exact test, corrected for multiple testing using the Benjamini-Hochberg method.

F,G, Western blot analysis to verify the depletion of PNPT1 upon siRNA treatment (F) and a representative immunofluorescence image of siPNPT1/siControl-treated control and W6A mutant MDA-MB-231 cells stained with J2 antibody (red). DAPI was used to mark the nucleus (blue) (G). The right-most panels show a magnification of the cells enclosed by the white boxes in the middle panels.

H, Model of AGO1x function. A complex of AGO1x with PNPT1 interacts with nuclear RNAs to prevent the accumulation of structured dsRNAs, supporting proliferation of breast cancer cells. Depletion of AGO1x leads to deleterious accumulation of these RNAs, which in turn lead to activation of interferon (IFN) response and apoptosis.

Table 1 Plasmids and oligonucleotides used in this study.

Modified from Addgene #6798,

Additional nucleotides appended to the stop codon in #6798: aggcagaacgctgttacctcactggatagaagaaagctttccaagccccaggagctgtgccacccaaatccagaggaagcaaggaggagggaggtggggtag. The stop codon of #6798 modified to tcc.

Table 2 | Antibodies used in this study.

Expanded View Figure Legends
Adipocyte extracellular vesicles carry enzymes and fatty acids that stimulate mitochondrial metabolism and remodeling in tumor cells. (2019)
Figure 5

Figure 5: Adipocyte EV modify melanoma mitochondrial dynamics, a process that promotes melanoma aggressiveness and is exacerbated by obesity

A) 1205Lu cells exposed to 3T3-F442A EV (3T3-EV) and treated, or not, with Mdivi-1, were stained with a MitoTracker probe, fixed and observed by confocal microscopy. Arrows indicate the presence of mitochondria in membrane protrusions. On the images to the right, the outline of cells is shown in dotted lines. Scale bars: 20µm. Right panel, quantification of the distance between mitochondria and nuclei (n=15).

B) Western blot analysis of the indicated mitochondrial fission proteins in melanoma cells treated, or not, with EV from 3T3-F442A adipocytes (3T3-EV). Tubulin (TUB) is used as a loading control.

C) 1205Lu cells were exposed to 3T3-F442A EV (3T3-EV) and treated, or not, with Mdivi-1. Cell migration was then evaluated in Boyden chamber assays (left panel; n=6). Right panel, 3T3-F442A EV were added to 1205Lu cells, which were immediately treated, or not, with Mdivi-1. Cell motility was then tracked by video microscopy (n = 40 cells per group) (right panel).

D) 1205Lu cells were transfected, or not, with 2 different siRNA targeted against DRP1 (siDRP1#1 or 2) or an untargeted siRNA (siUT). Top, 48h after transfection, protein extracts were prepared and DRP1 expression was evaluated by western blot. Bottom, 36h post transfection, cells were exposed to 3T3-F442A EV (3T3-EV) and cell motility was tracked by video microscopy (n = 40 cells per group).

E) 1205Lu cells were exposed to adipocyte EV from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD) and immediately treated, or not, with Mdivi-1. Cell motility was then tracked by video microscopy (n = 40 cells per group).

Data information: Bars and error bars represent means ± SEM; statistically significant by Student's t-test (A) and one-way ANOVA with post hoc Tukey's test (C-E) except (C, left panel) analyzed using Kruskal-Wallis with post-hoc Dunn's test. *P < 0.05, **P < 0.01. ***P < 0.001, ns: non-significant.
Adipocyte extracellular vesicles carry enzymes and fatty acids that stimulate mitochondrial metabolism and remodeling in tumor cells. (2019)
Figure 7

Figure 7: FA metabolism and mitochondrial dynamics are associated with melanoma aggressiveness

A) TCGA data were analyzed to reveal the effect of high mRNA levels of key FAO enzymes on the overall survival of patients with melanoma. Statistical significance of survival was evaluated with a Log-rank (Mantel-Cox) Test (n=449, 450 and 459 for HADHA, HADHB and ECHS1 respectively).

B) BODIPY staining of neutral lipid stores in the indicated melanoma cell lines, which present increasing degrees of aggressiveness (from left to right). DAPI was used to counterstain nuclei. Scale bar: 20µm.

C) FAO in the indicated melanoma cell lines was measured. Bars and error bars represent means ± SEM (n=5); statistically significant by one-way ANOVA with post hoc Tukey's test, ***P < 0.001. If not indicated, non-significant.

D) TCGA data were analyzed to reveal the effect of high mRNA levels of key actors in mitochondrial dynamics on the overall survival of patients with melanoma. Statistical significance of survival was evaluated with a Log-rank (Mantel-Cox) Test (n=459).

Expanded View Figure Legends
TOP3A amplification and ATRX inactivation are mutually exclusive events in pediatric osteosarcomas using ALT (2022)
Figure 1

Figure 1. High frequency of ALT in high-grade osteosarcomas

(A) C-circle assay for tumor samples. The presence of C-circles was tested in 22 osteosarcomas by Rolling Circle Amplification (RCA) assay using Φ29 DNA polymerase. Φ29 DNA polymerase negative controls and Φ29 DNA polymerase-based reactions starting respectively with 75, 37.5, and 18.75 ng of DNA were applied to dot blots and C-circles were detected by hybridization with a 32P-(CCCTAA)4 telomeric probe. U2OS and HeLa cells correspond to positive and negative control samples, respectively.

(B) Representative images of telomere FISH (red) and PML IF (green) colocalizations (APBs) in osteosarcoma tumor samples. DNA is counterstained blue with DAPI. APBs are indicated by white arrows. Scale bars are 5 μm.

(C) Representative image of TeSLA Southern Blot of two representative samples of ALT-positive (left) and telomerase-positive (right) osteosarcomas. Nine TeSLA PCRs (30 pg each reaction) were done for each DNA sample. DIG-labelled MW ladder has been added a posteriori.

(D) Plot of cumulative TeSLA fragment sizes (in kb) in ALT-positive (n=16) and ALT-negative (n=6) osteosarcomas. Points and error bars (± SEM) represent cumulative percentage of TeSLA fragments from all the TeSLA PCRs (n=9 per tumor sample) from all the patients in both groups (n=16 in ALT-positive group, and n=6 in ALT-negative group). The Kolmogorov-Smirnov test was applied to identify statistical differences in TeSLA fragments distributions (p=0.0016).

(E) Individual TeSLa fragment lengths in ALT-positive (n=16) and ALT-negative (n=6) osteosarcomas. Each dot represents a TeSLA fragment, bars represent the median value for individual patient's samples.
TOP3A amplification and ATRX inactivation are mutually exclusive events in pediatric osteosarcomas using ALT (2022)
Figure 2

Figure 2. ATRX-wt in expressed in ALT-positive high-grade osteosarcomas

(A) Oncoprint graph showing the distribution of mutations (green rectangles) and copy number variations (red rectangle, amplification; blue rectangle, copy loss) in all samples. Tumors are distributed according to ALT-positivity (ALT+) or -negativity (ALT-) and ATRX status (ATRX-mutated or ATRX-wt).

(B) Tissue sections of two representative high-grade osteosarcomas with HES (hematoxylin eosin saffron) staining (left panel), anti-ATRX (HPA064684) immunochemistry (middle panel), and anti-DAXX (HPA008736) immunochemistry (right panel), showing ATRX protein expression in two ALT-positive samples. In the top panel, intratumoral ATRX expression is negative (positive control osteoclasts are shown). In the bottom panel, intratumoral ATRX expression is high. Scale bars are 250 μm.

(C) STRING network showing mutated gene-clusters with related functions.
TOP3A amplification and ATRX inactivation are mutually exclusive events in pediatric osteosarcomas using ALT (2022)
Figure 3

Figure 3. TOP3A amplification and ATRX inactivation are mutually exclusive in ALT pediatric osteosarcoma

(A) Copy-number-alteration (CNA) profiles of osteosarcomas. Representation of the GISTIC analysis with false discovery rate (FDR (q-value)) for all cases (n=22; left panel), ALT-positive/ATRX-wt cases (n=11; middle panel), and ALT-positive/ATRX-mutated cases (n=5; right panel).

(B) Gain/amplification of TOP3A gene region (17p11) is a genomic signature of ATRX-wt osteosarcomas. For each ATRX-mutated (top part) and ATRX-wt (bottom part) case, from the left to the right, the chromosome 17 and regional genomic profiles were established, with CGH Analytics software. A focus was made within the genomic interval (17.25-19.11 Mb) of the short arm of chromosome 17 (hg38 human genome mapping; Build 38 from NCBI, December 2013 version) including TOP3. Color profiles corresponding to the different tumors are defined at the top of each group. Most ATRX-wt cases show gain or amplification of this region (bottom part), whereas no ATRX-mutated cases display this alteration (top part).

(C) Chromosome 17 copy number variation analysis showing amplification/gain of the TOP3A region that do not extend into the TP53 gene in ALT ATRX-wt tumors (left panel), and amplification of the TOP3A region for which TP53 region is at the edge of the amplification/gain in ALT-positive ATRX-wt tumors (right panel).
TOP3A amplification and ATRX inactivation are mutually exclusive events in pediatric osteosarcomas using ALT (2022)
Figure 4

Figure 4. TOP3A is overexpressed in ALT-positive/ATRX-wt osteosarcomas

(A) Volcano plot of the supervised analysis of mRNA expression profiles of osteosarcomas according to ALT-positive vs. ALT-negative samples, showing upregulated (in red) and downregulated (in green) genes. Twelve tumors had sufficient RNA quality to be analyzed: seven ALT-positive ATRX-wt, two ALT-positive/ATRX-mutated and three ALT-negative.

(B) TOP3A gene expression according to ALT and ATRX status: ALT+/ATRX-wt (n=7), ALT+/ATRX-mut (n=2) and ALT- (n=3). Box-and-whisker plot were defined with default parameters by median value (central band at the 50th percentile), interquartile ranges (IQR, box limited by 25th and 75th percentile) and whisker boundaries defined by minimum and maximum value. An ANOVA statistical test, and Tukey's range test were used to compare modalities.

(C) Normalized enrichment score bar plot of top 15 significant REACTOME gene sets according to ALT-positive vs. ALT-negative GSEA analysis. Ontologies associated with ALT-positivity are in red and those associated with ALT-negativity are in blue.
TOP3A amplification and ATRX inactivation are mutually exclusive events in pediatric osteosarcomas using ALT (2022)
Figure 5

Figure 5. ATRX is functional in ALT-positive ATRX-w cell lines and its ALT inhibitory function is counteracted by overexpression of TOP3A

(A) Western blot showing ATRX protein expression in nine osteosarcoma cell lines and HTC116 as positive control.

(B) Western blot showing TOP3A protein expression in nine osteosarcoma cell lines and NY siTOP3A as control.

(C) Telomere FISH (green), and PML (red) and TOP3A (purple) IF, showing TOP3A localization at APBs in both ATRX-wt and ATRX-mutated ALT-positive cells. TOP3A foci are indicated by white arrows. Scale bars are 5 μm.

(D) C-circle assay showing inverse effect of ATRX ectopic expression on C-circle levels in ATRX-mutated and ATRX-wt cell lines. Error bars represent the mean ± SEM from n = 2 experiments, n.s. = non-significant, *p < 0.05, Mann-Whitney test.

(E) C-circle assay showing rescue of ATRX ectopic expression by TOP3A overexpression in U2OS osteosarcoma cells Error bars represent the mean ± SEM from n = 2 experiments, n.s. = non-significant, *p < 0.05, Mann-Whitney test. Western blot and quantification of TOP3A expression is shown for U2OS cells overexpressing TOP3A.

(F) APB frequency and mean telomeric DNA intensity in APBs according to TOP3A overexpression and ATRX transient expression; n = 150 cells scored per treatment, n.s., non-significant, *p < 0.05; Mann-Whitney test.
TOP3A amplification and ATRX inactivation are mutually exclusive events in pediatric osteosarcomas using ALT (2022)
Figure 6

Figure 6. TOP3A inhibition affects ALT phenotype and leads to telomeric DNA damage

(A) Relative C-circle intensity according to TOP3A knock-down (KD) in ALT-positive ATRX-mutated or ATRX-wt cell lines. Error bars represent the mean ± SEM from n = 2 experiments.

(B) Representative images of telomeric DNA (green) and PML protein (red) colocalizations (APBs) in osteosarcoma and in vitro-immortalized ATRX-mutated or ATRX-wt cell lines according to TOP3A KD. Scale bars are 5 μm.

(C) Quantification of APB frequency in osteosarcoma and in vitro-immortalized ATRX-mutated or ATRX-wt cell lines according to TOP3A KD; Error bars represent the mean ± SEM from n = 3 experiments, n = 150 cells scored per treatment, n.s., non-significant; Mann-Whitney test.

(D) Effect of TOP3A KD on Telomere Dysfunction-Induced Foci (TIFs) in osteosarcoma and in vitro-immortalized cell lines; Error bars represent the mean ± SEM from n = 3 experiments, n = 150 cells scored per treatment, *p < 0.05, **p < 0.005; Mann-Whitney test.

(E) Distributions of TeSLA fragments in osteosarcoma and in vitro-immortalized ATRX-mutated or ATRX-wt cell lines according to TOP3A KD (left panel; each dot represents a TeSLA fragment), and representative TeSLA Southern Blot image for NY (right panel).