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. 2023 Sep 22;14:5922. doi: 10.1038/s41467-023-41545-7

Fig. 2. Expression of UBB+1 induces Amyloid beta (Aβ) and phosphorylated tau (p-tau) aggregates in a 3D human neural culture system.

Fig. 2

a Schematic of UBB+1 expressing 3D system derived from ReN VM cells. Image was created using BioRender software. b Representative immunofluorescence images of 6-week-old differentiated control, APPSL-PSNE1ΔE9 (FAD) and UBB+1 expressing 3D cultures, showing Aβ-positive-aggregates in white arrows, indicating that they are extracellular deposits. c Quantification of the mean total number of extracellular Aβ deposits in 6-week-old control, FAD, and UBB+1 cultures [n = 3 biologically independent samples, the whole well was counted] (FAD/ Ctrl. p = 0.000029; UBB+1 /Ctrl. p = 0.00016). d Immunoblot analysis of SDS-solubilized aggregates isolated from 6-week-old -differentiated control, FAD, and UBB+1 cultures, showing Aβ oligomers. β-tubulin III levels were used as loading control. e ELISA of Aβ42 and Aβ40 in conditioned media collected from 3D 6-week-old control, FAD, and UBB+1 cultures. Aβ42/Aβ40 ratio is displayed [n = 3 biologically independent samples] (FAD/Ctrl. p = 0.007; UBB+1 /Ctrl. p = 0.04). f ELISA of Aβ42 in whole lysates of 3D 8-week-old control, FAD, and UBB+1 cultures. Results are displayed as Aβ42 normalized to total protein [n = 3 biologically independent samples] (FAD/ Ctrl. p = 0.014; UBB+1 /Ctrl. p = 0.043). g Representative immunohistochemistry images of 3D 8-week-old FAD and UBB+1 cultures stained with anti- p-tau. Cells displaying increased p-tau staining are marked by black arrowheads. h Quantification of the mean number of p-tau positive-stained cells [n = 3 biologically independent samples, whole well was counted] (FAD/ Ctrl. p = 0.004; UBB+1 /Ctrl. p = 0.007). i Sarkosyl-soluble fractions extracted from 3D FAD and UBB+1 -8-week-old thick cultures immunoblotted for tau or p-tau. Calnexin levels were used as loading control. j Representative image of Cryo-TEM of 14-week-old UBB+1 Sarkosyl-insoluble fraction showing specific binding of tau antibody to aggregates, visualized with 5 nm nano-gold particles. P-values were determined by unpaired two-tailed Student’s t-test. Error bars represent ± s.d. Images are representative of three independent samples. All experiments were repeated at least twice. Scale bars: 20 µm (b), 50 µm (g), and 100 nm (j).