Fig. 7: FUSIBB lowers endogenous FUS expression and is not toxic to cells.
From: Engineered NLS-chimera downregulates expression of aggregation-prone endogenous FUS
A Immunofluorescence images of HEK293 cells transfected with FLAG-tagged FUSIBB (or GFP). NT = non-transfected. Solid lines circle transfected cells, and dashed lines circle non-transfected cells. The experiment was repeated at least three times with similar results. Scale bars = 20 µm. B Quantification of endogenous FUS fluorescence intensity shown in A. Mean and SD of n = 304 for GFP non-transfected cells, n = 285 for GFP transfected cells, n = 708 for FUSIBB non-transfected cells, and n = 378 for FUSIBB transfected cells. ****padj < 0.0001 by one-way ANOVA Sidak’s multiple comparisons test. Data from one representative experiment is shown. Experiments were repeated at least three times with similar results. C Western blot of HEK293 cells transfected with FLAG-tagged FUSIBB (or mClover). NT = non-transfected. D Quantification of Western blot images in C. Mean and SEM of n = 3 independent experiment. Two-tailed, unpaired t test. E mRNA level of endogenous FUS normalized to β-Actin in GFP or FUSIBB transfected U2OS cells, measured by RT-qPCR. Mean and SEM of n = 4 independent experiments. Two-tailed, unpaired t test. F Normalized anisotropy of fluorescein-labeled BDNF RNA with different concentrations of MBP-tagged WT FUS or FUSIBB. Mean and SEM of n = 3 independent experiments. Solid lines represent the fitted curve. G The dissociation constant, Kd, fitted from the dose-response curves in F. Mean and SEM of n = 3 independent experiments. ns = non-significant (p > 0.05) by two-tailed, unpaired t test. H Representative Western blot image of the doxycycline-inducible FUSIBB cell lysates. M = molecular weight marker. β-Actin = loading control. The experiment was repeated at least twice with similar results. I Cell viability assay of doxycycline-inducible FUSIBB cells. Cell viability was estimated based on resazurin (non-fluorescent) conversion to resorufin (fluorescent). Mean and SEM of n = 4 wells from a representative experiment. The experiments were repeated at least 3 times with similar results. ***p < 0.001 and ****p < 0.0001 by two-way ANOVA Dunnett’s multiple comparisons test with Geisser-Greenhouse correction. J The model for FUSIBB expression replacing the endogenous FUS. Endogenous FUS is prone to aggregation under stress. FUSIBB can maintain nuclear localization and downregulate endogenous FUS expression, suggesting its potential as a therapeutic agent to replace disease-causing FUS. Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.Source data are provided as a Source Data file.
