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: For FUS knockdown, 150,000 HEK293 cells in a 6‐well plate were transfected with 25 nM FUS SMARTpool: siGENOME siRNA (Dharmacon, Canada, L‐009497‐00‐0005) or ON‐TARGET plus Nontargeting pool siRNAs (Dharmacon, D‐001810‐10‐05) with DharmaFECT one transfection reagent (Dharmacon, T‐2001–02) according to the manufacturer’s protocol.

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: For FUS knockdown, 150,000 HEK293 cells in a 6‐well plate were transfected with 25 nM FUS SMARTpool: siGENOME siRNA (Dharmacon, Canada, L‐009497‐00‐0005) or ON‐TARGET plus Nontargeting pool siRNAs (Dharmacon, D‐001810‐10‐05) with DharmaFECT one transfection reagent (Dharmacon, T‐2001–02) according to the manufacturer’s protocol.

Techniques: Concentration Assay, Recombinant, Western Blot