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Fig. 7 | Cell & Bioscience

Fig. 7

From: FBXL17/spastin axis as a novel therapeutic target of hereditary spastic paraplegia

Fig. 7

The pathogenicity of SPAST-Y52C was rescued by the inhibition of SCFFBXL17 in a 3D model. A Immunostaining of GFP and acetylated α-tubulin in ReNcell CX transfected with GFP-empty, WT and Y52C of GFP-SPAST-M1. Fluorescence intensities of acetylated α-tubulin quantified with ImageJ software. Scale bar: 50 μm. B After neuronal differentiation for 2 d, immunostaining of GFP and Tau in ReNcell CX transfected with GFP-empty, WT and Y52C of GFP-SPAST-M1. Scale bar: 20 μm. Quantification of mean axonal length in each group. C ReNcell CX were transduced with each indicated lentiviral vector (LV, with 5 MOI) and transferred to ultra-low binding well plates. Cells were cultured under orbital shaking conditions for 4 d, and observed under a microscope using bright field and GFP filter (Scale bar: 100 μm). D The neurospheres’ diameter was measured and quantified with ImageJ software. E The protein expression of SPAST-WT or Y52C under the same conditions in (C) F After transduction with each indicated lentiviral vector (5 MOI), ReNcell CX were cultured under orbital shaking for 4 d and then another 4 d under differentiation conditions with or without 5 μM MLN4924. Cells were observed under a microscope using bright field and a GFP filter (Scale bar: 100 μm). G The neurospheres’ diameter was measured and quantified with ImageJ software. H The protein expression of SPAST, MAP2 and GFAP under the same conditions in (F). MAP2 was used as a mature neural marker, and GFAP was used as an astrocyte marker. All experiments were performed in triplicate, data are expressed as the mean of three samples with SD, and results are representative of three independent experiments. (*P < 0.05; ** P < 0.01; ns, not significant)

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