altFUS, a novel endogenous mitochondrial protein 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).Figure 2A, 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 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. AltFUS (arrow) endogenous expression in the motor cortex of three C9orf72 and three sporadic ALS patients (D) (representative image from n=3). The asterisk indicates a protein species detected with the anti-altFUS antibody specifically in the brain. AltFUS (arrow) endogenous expression 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 Tomm20 (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 (Flag). 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 siFUS 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 (Tomm20 in red) in mock and altFUS-Flag (green) transfected HeLa cells (n=3). The white scale bar corresponds to 10μm. K, Proportion of tubules and globules in the mitochondrial network of mock HeLa cells and HeLa cells transfected with altFUS-Flag 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 25th and 75th percentiles, with the median marked. The whiskers correspond to the 5th and 95th percentiles.