Journal: International Journal of Medical Sciences

Article Title: Bioinformatics Analyses of Potential miRNA-mRNA Regulatory Axis in HBV-related Hepatocellular Carcinoma

doi: 10.7150/ijms.50126

Figure Lengend Snippet: Expression level and prognosis value of hub genes. (A) Protein expressions of hub genes were up regulated in HCC tissues. Images were taken from the Human Protein Atlas ( http://www.proteinatlas.org ) online database; (B) The mRNA expressions of hub genes were up regulated. Data were taken from the GEPIA database. (C) Kaplan-Meier analysis of hub genes for patients with HCC. Red boxes and grey boxes represent the mRNA levels of tumor and nomal tissue, respectively. * P < 0.05.

Article Snippet: Protein expressions of hub genes in normal and HCC tissues were analyzed in The Human Protein Atlas , .

Techniques: Expressing

Journal: EMBO Reports

Article Title: The FUS gene is dual‐coding with both proteins contributing to FUS ‐mediated toxicity

doi: 10.15252/embr.202050640

Figure Lengend Snippet: A FUS gene bicistronic annotation, with the canonical FUS CDS (in blue, +1 frame) and altFUS CDS (in green, +2 frame), represented on the FUS canonical transcript ( ENST00000254108 or NM_004960 ). Sequence length proportions are respected, and the scale bar corresponds to 300 nucleotides. B Genome browser view of FUS gene. The five most abundant transcripts in the brain are shown in the “Transcript” track. Transcripts predicted coding by the OpenProt resource are coloured in blue, and in grey if predicted non‐coding. The “Protein” track contains all predicted protein products. The known FUS protein (ENSP00000254108) and its isoform (ENSP00000369594) are coloured in green. The novel‐predicted altFUS protein (IP_243680) and its isoform (IP_243691) are coloured in red. C PhyloP nucleotidic conservation scores are represented in grey across the FUS mRNA (ENST00000254108). The noise reduction after FFT (fast Fourier transformation) is outlined in blue. The average PhyloP score on the bicistronic and the monocistronic region are represented as dotted red lines. The position of the FUS CDS is represented by a blue rectangle and that of altFUS CDS by a green rectangle. D Alignment (Clustalω) of altFUS protein sequences in human ( Homo sapiens ), chimpanzee (Chimp.— Pan troglodytes ), rat ( Rattus norvegicus ), mouse ( Mus musculus ) and dog ( Canis lupus familiaris ). Residues are coloured based on their identity across species, from white (not conserved) to red (conserved in all species). E R ibo ‐seq data over the FUS gene from the GWIPS portal. Initiating ribosome reads are indicated by blue bars, and elongating ribosomes footprints are indicated by the blue curve. The graph captures the beginning of the FUS gene with FUS and altFUS methionines indicated by blue arrows. The genomic positions are indicated relative to the start of exon 1. F, G Genome browser view of FUS gene, centred on altFUS. The “Transcript” track contains the beginning of the canonical FUS transcript ( ENST00000254108 ) in blue. The “Protein” track contains the beginning of the FUS protein (green) and the whole altFUS protein (red). In F, the “Peptide” track contains all the peptides identified by the OpenProt resource using the classical spectrum‐centric approach. The peptides sequences are indicated and are unique to altFUS or its isoform (see Fig for an example spectrum). In G, the “Peptide” track contains all the peptides identified by a peptide‐centric approach. The peptides indicated matched better to at least one spectrum than any known protein and are coloured in yellow if they matched better than any known protein with any PTM (see Fig for an example spectrum). The peptides sequences are indicated and are unique to altFUS or its isoform.

Article Snippet: Human tissue lysates for altFUS endogenous expression were purchased from Zyagen Laboratories (San Diego, California, USA).

Techniques: Sequencing, Transformation Assay

Journal: EMBO Reports

Article Title: The FUS gene is dual‐coding with both proteins contributing to FUS ‐mediated toxicity

doi: 10.15252/embr.202050640

Figure Lengend Snippet: A AltFUS is encoded within an alternative open reading frame (ORF) overlapping the FUS canonical CDS. When read in the + 1 frame, the FUS mRNA (here ENST00000254108 ) codes for a 526 amino acid protein (highlighted in blue), named FUS. In the + 2 frame, the FUS mRNA contains a second ORF (highlighted in green) that codes for a novel 170 amino acid protein, named altFUS. The two proteins are not isoforms. B GTEx portal data on FUS mRNA expression in the brain and nerves are shown in blue coloured scale (TPM = transcripts per million). Transcripts are identified with Ensembl accessions (the number after the dot corresponds to the version used in the analysis) and are quantified across 14 tissues. The five transcripts framed in red share 85% of all mRNA expression level. C Table compiling the protein information relayed by Ensembl and OpenProt resources for the five transcripts highlighted in panel B. The FUS protein is highlighted in blue. The altFUS protein is highlighted in green. D–E Alignment (Clustalω) of protein sequences for FUS (ENSP00000254108) and its isoform (ENSP00000369594) in panel D (blue) and altFUS (IP_243680) and its isoform (IP_243691) in panel E (green). The residues are coloured based on their degree of identity. F Heatmap of altFUS protein sequences identity across 84 species (see Table and Appendix Fig ). Primates, rodents and mammals in general display strong protein conservation. The sequence identity is coloured from blue (0%) to red (100%).

Article Snippet: Human tissue lysates for altFUS endogenous expression were purchased from Zyagen Laboratories (San Diego, California, USA).

Techniques: Expressing, Sequencing

Journal: EMBO Reports

Article Title: The FUS gene is dual‐coding with both proteins contributing to FUS ‐mediated toxicity

doi: 10.15252/embr.202050640

Figure Lengend Snippet: R ibo ‐seq data over the mouse FUS gene from the GWIPS portal ( Mus musculus ). Initiating ribosome reads are indicated by green bars, and elongating ribosomes footprints are indicated by the green curve. The graph captures the beginning of the FUS gene with FUS and altFUS methionines indicated by green arrows. The genomic positions are indicated relative to the start of exon 1. MS/MS spectrum confidently mapped to a peptide unique to altFUS by the OpenProt resource. Peaks are represented by their mass over charge ratios (m/z) and their intensity relative to the highest (relative intensity). The y ions are coloured in blue, the b ions in red and the unannotated peaks appear in grey. The charge state and m/z error of the matched peptide are indicated on the top right corner of the spectrum. The original study, file and spectrum number are indicated below the graph. Comparison of one MS/MS spectrum confidently mapped to a peptide unique to altFUS (left graph) and its best possible annotation with any known protein with any post‐translational modification (PTM—right graph). Peaks are represented by their mass over charge ratios (m/z) and their intensity relative to the highest (relative intensity). The y ions are coloured in blue, the b ions in red and the unannotated peaks appear in grey. The charge state and m/z error for each of the matched peptides are indicated on the top right corner of each panel. The original study, file and spectrum number are indicated below the graph. On the top of each panel is indicated the peptide matched to the spectrum and the corresponding protein and PTMs.

Article Snippet: Human tissue lysates for altFUS endogenous expression were purchased from Zyagen Laboratories (San Diego, California, USA).

Techniques: Tandem Mass Spectroscopy, Comparison, Modification

Journal: EMBO Reports

Article Title: The FUS gene is dual‐coding with both proteins contributing to FUS ‐mediated toxicity

doi: 10.15252/embr.202050640

Figure Lengend Snippet: A Expression of untagged versions of both FUS and altFUS proteins from transfection of the FUS cDNA in HEK293 cells by Western blot, and expression of FUS with the monocistronic construct FUS (Ø) (representative image from n = 3). The slight decrease in endogenous FUS expression upon altFUS over‐expression (third lane) was not significant (see Appendix Fig F for quantification, P = 0.255, n = 3). B AltFUS endogenous expression in HEK293 cells using a siRNA targeting FUS mRNA as negative control and over‐expression of altFUS CDS as positive control (representative image from n = 3). For the mock, siCTRL and siFUS conditions, 100 μg of total protein was loaded, when only 15 μg of total proteins was loaded for the altFUS over‐expression condition. C AltFUS (arrow) endogenous expression in human tissues (brain, muscles and kidney—100 μg), in HEK293 and HeLa‐cultured cells (100 μg) and using the over‐expression of altFUS CDS in HEK293 cells (50 μg) as positive control (representative image from n = 3). The asterisk indicates a protein species detected with the anti‐altFUS antibody specifically in the brain. D, E AltFUS (arrow) endogenous expression in the motor cortex of three C9orf72 and three sporadic ALS patients (D) or in iPSC‐derived motor neurons of three lines from controls and from ALS patients (E) (representative image from n = 3). The asterisk indicates a protein species detected with the anti‐altFUS antibody specifically in the brain. F Images by confocal microscopy of altFUS‐ flag (green) in HeLa cells, using T omm 20 (red) as a mitochondrial marker (representative image from n = 3, Pearson’s correlation r = 0.92). Mock‐transfected cells were identically stained, highlighting the specificity of the observed altFUS signal (F lag ). The white scale bar corresponds to 10 μm. G AltFUS‐ flag enrichment in mitochondrial extracts from transfected HEK293 cells (representative image from n = 3) with mtHsp70 used as a mitochondrial marker (WCL = whole cell lysate, Mito = mitochondrial extract). H AltFUS mitochondrial expression in untagged altFUS‐transfected HEK293 cells following fractionation (representative image from n = 3), with Tubulin as a marker of the cytosolic fraction and VDAC as a marker of the mitochondrial fraction (WCL = whole cell lysate, Cyto = cytosol fraction, Mito = mitochondrial fraction). I Endogenous altFUS mitochondrial expression in HEK293 cells following fractionation (representative image from n = 3), with Tubulin as a marker of the cytosolic fraction and VDAC as a marker of the mitochondrial fraction. We used si FUS ‐transfected cells as a negative control and altFUS‐transfected cells as a positive control for altFUS expression (WCL = whole cell lysate, Cyto = cytosol fraction, Mito = mitochondrial fraction). J Representative images of the mitochondrial network (T omm 20 in red) in mock and altFUS‐F lag (green)‐transfected HeLa cells ( n = 3). The white scale bar corresponds to 10 μm. K Proportion of tubules and globules in the mitochondrial network of mock HeLa cells and HeLa cells transfected with altFUS‐ flag (see Appendix Fig A). Quantification was done over a minimum of 100 cells across a technical duplicate per independent experiments ( n = 3, i.e. a minimum of 300 cells per biological conditions, p ‐value < 0.001, Mann–Whitney U test). The boxes extend to the 25 th and 75 th percentiles, with the median marked. The whiskers correspond to the 5 th and 95 th percentiles.

Article Snippet: Human tissue lysates for altFUS endogenous expression were purchased from Zyagen Laboratories (San Diego, California, USA).

Techniques: Expressing, Transfection, Western Blot, Construct, Over Expression, Negative Control, Positive Control, Muscles, Cell Culture, Derivative Assay, Confocal Microscopy, Marker, Staining, Fractionation, MANN-WHITNEY

Journal: EMBO Reports

Article Title: The FUS gene is dual‐coding with both proteins contributing to FUS ‐mediated toxicity

doi: 10.15252/embr.202050640

Figure Lengend Snippet: Estimation of the concentration of GFP‐FUS and GST‐altFUS recombinant proteins (10 μL loaded each). A commercial LSD1 recombinant protein diluted at 0.2 μg/μL was used as standard. The concentration for GFP‐FUS was evaluated to 0.06 μg/μL and that of GST‐altFUS was evaluated to 0.07 μg/μl. Western blot of human brain lysates spiked with GFP‐FUS (last lane). The recombinant protein alone (first lane) and a whole cell lysate of HEK293 cells were used as size controls. The bands corresponding to the recombinant GFP‐FUS and to the endogenous FUS proteins are indicated on the left. Immunoblot: anti‐FUS (Abcam, ab84078). Western blot of human brain lysates spiked with GST‐altFUS (last lane). The recombinant protein alone (first lane) and a whole cell lysate of HEK293 cells were used as size controls. The bands corresponding to the recombinant GST‐altFUS and to the endogenous altFUS proteins are indicated on the left. The asterisk indicates a protein species detected with the anti‐altFUS antibody specifically in the brain (see Fig ). Immunoblot: custom anti‐altFUS.

Article Snippet: Human tissue lysates for altFUS endogenous expression were purchased from Zyagen Laboratories (San Diego, California, USA).

Techniques: Concentration Assay, Recombinant, Western Blot

Journal: EMBO Reports

Article Title: The FUS gene is dual‐coding with both proteins contributing to FUS ‐mediated toxicity

doi: 10.15252/embr.202050640

Figure Lengend Snippet: Representative traces of TMRE fluorescence measured by flow cytometry in mock‐transfected cells and cells over‐expressing the bicistronic FUS‐R495x or the monocistronic FUS (Ø) ‐R495x constructs ( n = 4, minimum of 50 000 live cells per independent replicates). Mean fluorescence intensity of mock‐transfected cells treated with a decoupling agent, FCCP, is indicated by a grey dotted line. Mean TMRE fluorescence intensity measures in mock‐transfected cells, cells over‐expressing altFUS, FUS, FUS (Ø) , FUS‐R495x or FUS (Ø) ‐R495x, or mock‐transfected cells treated with FCCP across 4 independent experiments (n = 4, also see Appendix Fig D, mean ± SD). Statistical significance is relative to the mock condition unless otherwise indicated (*** P < 0.001, **** P < 0.0001, n.s. = non‐significant, two‐way ANOVA with Tukey’s multiple comparison correction). Representative image by stimulated emission depletion microscopy (STED) of altFUS‐F lag (green) localization within mitochondria (T omm 20 marker in red). The white bar across the mitochondria represents the region of interest quantified in panel D. The white scale bar corresponds to 3 μm. Relative fluorescence histogram for altFUS‐F lag and T omm 20 across the region of interest highlighted by a white line on panel C. Scatter plot of the proteins identified by AP‐MS (see Appendix Fig ) indicating their enrichment (fold change over control) and their SAINT probability score. Proteins above the 0.8 threshold (grey line) are indicated in black, others in grey. AltFUS is indicated in red (bait), and preys known to regulate the autophagy or the cellular stress response are indicated in green. Subcellular localizations of proteins identified by AP‐MS from panel E (see Appendix Fig ). Enrichment of biological processes in altFUS‐interacting proteins compared with the human mitochondrial proteome (Fisher’s exact test with FDR < 0.1%). The number of proteins identified in each GO term is indicated next to the corresponding bar.

Article Snippet: Human tissue lysates for altFUS endogenous expression were purchased from Zyagen Laboratories (San Diego, California, USA).

Techniques: Fluorescence, Flow Cytometry, Transfection, Expressing, Construct, Comparison, Microscopy, Marker, Protein-Protein interactions, Control

Journal: EMBO Reports

Article Title: The FUS gene is dual‐coding with both proteins contributing to FUS ‐mediated toxicity

doi: 10.15252/embr.202050640

Figure Lengend Snippet: A Images by confocal microscopy of mCherry‐GFP‐LC3 signal in HeLa cells across biological conditions: untreated mock, bafilomycin‐treated mock, altFUS, FUS, FUS (Ø) , FUS‐R495x and FUS (Ø) ‐R495x (representative images of n = 3). The altFUS signal (white) is shown as an inset in the top or bottom left of the left panels. The white scale bar corresponds to 10 μm, and the zoomed in region (right panels) is delimited by a white box. B LC3‐II accumulation after bafilomycin treatment from mock, altFUS, FUS, FUS (Ø) , FUS‐R495x, FUS (Ø) ‐R495x transfected cells and FUS (Ø) ‐R495x and altFUS co‐transfected cells across 3 independent experiments ( n = 3, mean ± SD). The quantification corresponds to the treated/untreated ratio of LC3‐II abundance (see Appendix Fig B for a representative image). Statistical significance is relative to the mock condition unless otherwise indicated (**** P < 0.0001, *** P < 0.001, ** P < 0.01, n.s. = non‐significant, two‐way ANOVA with Tukey’s multiple comparison correction). C Images by confocal microscopy of altFUS ( flag ‐tagged—white), FUS (GFP‐tagged—green) and TDP‐43 (red) signals in HeLa cells transfected with the bicistronic GFP‐FUS (F lag ) ‐R495x or the monocistronic GFP‐FUS (Ø‐ flag ) ‐R4955x constructs, or co‐transfected with the monocistronic GFP‐FUS (Ø‐F lag ) ‐R495x and altFUS‐F lag constructs (representative images from n = 3). The white scale bar corresponds to 10 μm. D, E Quantification of FUS cytoplasmic granules, number (D) and area (μm 2 ) (E) in cells over‐expressing the bicistronic (+) or monocistronic (−) construct for FUS, FUS‐G156E, FUS‐R495x, FUS‐K510E, FUS‐Q519x, FUS‐Q519I‐fs527x, FUS‐R521C and FUS‐P525L. Statistical comparisons are made between bicistronic and monocistronic versions of each construct ( n = 3—biological replicates, mean ± SD, **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, n.s. = non‐significant, one‐way ANOVA test with Sidak’s multiple comparison).

Article Snippet: Human tissue lysates for altFUS endogenous expression were purchased from Zyagen Laboratories (San Diego, California, USA).

Techniques: Confocal Microscopy, Transfection, Comparison, Construct, Expressing

Journal: EMBO Reports

Article Title: The FUS gene is dual‐coding with both proteins contributing to FUS ‐mediated toxicity

doi: 10.15252/embr.202050640

Figure Lengend Snippet: A Images by confocal microscopy of altFUS (F lag ‐tagged—white), FUS (GFP‐tagged—green) and TIA‐1 (red) signals in HeLa cells transfected with the bicistronic GFP‐FUS (F lag ) ‐R495x or the monocistronic GFP‐FUS (Ø‐ flag ) ‐R495x constructs, or co‐transfected with the monocistronic GFP‐FUS (Ø‐F lag ) ‐R495x and altFUS‐F lag constructs (representative images from n = 3). The white scale bar corresponds to 10 μm. B–D Images by confocal microscopy of altFUS(F lag ‐tagged—white), FUS (GFP‐tagged—green) and TIA‐1 (red) signals in HeLa cells transfected with the bicistronic constructs (B), monocistronic constructs (C) or co‐transfected with altFUS‐F lag and the monocistronic constructs (D) of 6 ALS‐associated mutants: FUS‐G156E, FUS‐K510E, FUS‐Q519x, FUS‐Q519I‐fs527x, FUS‐R521C and FUS‐P525L (representative images from n = 3). The white scale bar corresponds to 10 μm.

Article Snippet: Human tissue lysates for altFUS endogenous expression were purchased from Zyagen Laboratories (San Diego, California, USA).

Techniques: Confocal Microscopy, Transfection, Construct

Journal: EMBO Reports

Article Title: The FUS gene is dual‐coding with both proteins contributing to FUS ‐mediated toxicity

doi: 10.15252/embr.202050640

Figure Lengend Snippet: A Crossbreeding strategy for Drosophila generation using the Elav‐GeneSwitch‐GAL driver as an inducible expression system specific to the motor neurons. B FUS and altFUS expression in mCherry (control), altFUS, FUS, FUS (Ø) , FUS‐R495x or FUS (Ø) ‐R495x expressing Drosophila from the control F1 and the RU‐486‐treated F1 (see panel A) at 1, 10 or 20 days post‐induction (representative image from n = 3). C–E Locomotion assay represented by the percentage of climbing success in control and RU‐486‐treated transgenic Drosophila expressing mCherry or altFUS (C), the bicistronic FUS or the monocistronic FUS (Ø) (D), and the bicistronic FUS‐R495x or the monocistronic FUS (Ø) ‐R495x (E) at days 1, 10 and 20 post‐induction. Statistical comparisons were made between each population ( n = 4—biological replicates). Indicated significance is between the monocistronic and the bicistronic transgenic flies of the RU‐486‐treated population (mean ± SD, n.s. = non‐significant, * P < 0.05, *** P < 0.001, two‐way ANOVA with Tukey’s multiple comparison correction).

Article Snippet: Human tissue lysates for altFUS endogenous expression were purchased from Zyagen Laboratories (San Diego, California, USA).

Techniques: Expressing, Control, Transgenic Assay, Comparison

Journal: EMBO Reports

Article Title: The FUS gene is dual‐coding with both proteins contributing to FUS ‐mediated toxicity

doi: 10.15252/embr.202050640

Figure Lengend Snippet: Graphical representation of FUS synonymous mutations (yellow) found in ALS patients. The canonical FUS mRNA is represented in dark blue ( ENST00000254108 or NM_004960 ). The FUS protein‐coding sequence is indicated in light blue, and the altFUS protein‐coding sequence is indicated in green. Images by confocal microscopy of TDP‐43 (white), FUS (GFP‐tagged—green) and altFUS (F lag ‐tagged—red) in HeLa cells over‐expressing GFP‐FUS (F lag) , GFP‐FUS (P31L‐F lag) ‐s44=, GFP‐FUS (A38V‐F lag) ‐g51=, GFP‐FUS (A46V‐F lag) ‐g59 = and GFP‐FUS (R64P‐F lag) ‐s77= ( representative images from n = 3). White arrows indicate some TDP‐43 aggregates. The white scale bar corresponds to 10 μm. Quantification of cells with TDP‐43 aggregates in HeLa cells (see panel B). The data are represented as the fold change compared with the GFP‐FUS (F lag ) expressing cells. Statistical significance is indicated above the bars ( n = 3—biological replicates, mean ± SD, **** P < 0.0001, two‐way ANOVA with Tukey’s multiple comparison correction).

Article Snippet: Human tissue lysates for altFUS endogenous expression were purchased from Zyagen Laboratories (San Diego, California, USA).

Techniques: Sequencing, Confocal Microscopy, Expressing, Comparison

Journal: EMBO Reports

Article Title: The FUS gene is dual‐coding with both proteins contributing to FUS ‐mediated toxicity

doi: 10.15252/embr.202050640

Figure Lengend Snippet: Images by confocal microscopy of altFUS (FLAG‐tagged—green) and mitochondria (T omm 20 marker, red) in HeLa cells over‐expressing the empty vector (mock), altFUS‐F lag , altFUS‐P31L‐F lag , altFUS‐A38V‐F lag , altFUS‐A46V‐F lag or altFUS‐R64P‐F lag constructs (representative images from n = 3). The white scale bar corresponds to 10 μm. Deconvolution over a maximum of 30 iterations on the green and red channels was performed for the zoomed in pictures. The white scale bar corresponds to 1 μm. Images by confocal microscopy of endogenous FUS (red) and altFUS (F lag ‐tagged—green) in HeLa cells over‐expressing the empty vector (mock), altFUS‐F lag , altFUS‐P31L‐F lag, altFUS‐A38V‐F lag, altFUS‐A46V‐F lag or altFUS‐R64P‐F lag constructs (representative images from n = 3). The white scale bar corresponds to 10 μm.

Article Snippet: Human tissue lysates for altFUS endogenous expression were purchased from Zyagen Laboratories (San Diego, California, USA).

Techniques: Confocal Microscopy, Marker, Expressing, Plasmid Preparation, Construct