Nature Biotechnology: doi: /nbt Supplementary Figure 1
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1 Supplementary Figure 1 Representative images of an in vitro comparison of several AAV serotypes regarding egfp expression in cochlear explants of CBA/CaJ mice. (A-F): Results after incubation at equal doses of genome containing (GC) particles for 48 hours. Scale bar = 200 µm. (G-J): Mean indicated by horizontal bar. Each condition had minimally N=3 for 48h, and N=2 for 48h+5d unless otherwise noted.
2 Supplementary Figure 2 egfp expression scoring system. egfp expression in supporting and limbus cells following AAV exposure of cochlear explants was assessed using a qualitative scoring system ranging from 0 (D, H) (lowest expression) to 3 (A, E) (highest level of expression). Representative images illustrate the range of expression both in terms of intensity and number of transduced cells with 0 representing no noted expression (D, H), 1 select number of cells expressing dimly (C, G), 2 low to moderate levels of expression in significant numbers of cells per microscopic field (B, F), and 3 high percentage of cells expressing egfp at levels ranging from moderate to high (A, E). Scale bar for all images as in A (for A-D) and E (for E-H) = 20 μm.
3 Supplementary Figure 3 egfp expression in limbus, supporting cells, and spiral ganglion neurons (SGNs) in cochlear explants of C57BL/6 mice. Expression in limbus and supporting cells was assessed using an egfp scoring system detailed in Supplementary Fig. 2. SGN transduction was evaluated by egfp-positive cell counts per microscopic field. Mean indicated by horizontal bar. Each condition had minimally N=3 for the 48h, and N=2 for the 48h+5d unless otherwise noted.
4 Supplementary Figure 4 egfp expression in limbus, supporting cells, and spiral ganglion neurons (SGNs) in cochlear explants of CBA/CaJ mice. Expression in limbus and supporting cells was assessed using an egfp scoring system detailed in Supplementary Fig. 2. SGN transduction was evaluated by egfp-positive cell counts per microscopic field. Mean indicated by horizontal bar. Each condition had minimally N=3 for the 48h, and N=2 for the 48h+5d unless otherwise noted.
5 Supplementary Figure 5 Bilateral cochlear transduction from base to apex in mouse cochleas with Anc80. Mice injected at P1 were sacrificed and cochleas were evaluated for egfp transgene expression 30 days following injection on histological section stained for TuJ1 (red) and Myo7A (blue). Efficient Anc80 transduction was observed in the injected cochlea extending up to apex (A/F), however also in the contralateral uninjected ear (B-E=from apex to base). (G) Close-up image of egfppositive and TuJ1-positive spiral ganglion neurons (SGNs). (H) Reconstructed 3D image for SGN evaluation of Anc80 transduction. Scale bars 100 µm (A-E) and 20 µm (F/G).
6 Supplementary Figure 6 Expression in the central nervous system and Anti-AAV Neutralizing Antibody (NAB) titers. (A) Axial section of a mouse brain after unilateral cochlear injection with Anc80. (B) Predominant expression in the cerebellum, in particular Purkinje cells (white arrowheads). Scale bars 1 mm (A) and 300 µm (B). (C) Anti-AAV Neutralizing Antibody (NAB) titers in serum and Cerebrospinal Fluid (CSF) in uninjected (n=4 for CSF, n=2 for serum) and Anc80 RWM-injected animals (n=6 for CSF, n=2 for serum). Titers reflect the dilution of serum or CSF at which 50% inhibition of transduction was observed in the NAB assay. Due to sample volume limitations, the limit of sensitivity for serum NAB was 1/4 and 1/52.5 for CSF.
7 Supplementary Figure 7 Vestibular function following Anc80 cochlear transduction. Mice were injected at P1 with Anc80.CMV.eGFP via the RWM and evaluated for expression and balance function on the rotarod device. (A) Expression of egfp (green) in the vestibular tissue is detected via confocal microscopy with immunofluorescent staining for Myo7A (red). (B) Rotarod data revealed no difference between injected and uninjected controls. The mean time until the mice fell off the device +/- s.d. is plotted. N=3 animals, 5 trials each (injected) and 2 animals 5 trials each (control). Scale bar = 50 µm.
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Supplementary Figure 1.
Supplementary Figure 1. Increased β cell mass and islet diameter in βtsc2 -/- mice up to 35 weeks A: Reconstruction of multiple anti-insulin immunofluorescence images showing differences in β cell mass
(a) Significant biological processes (upper panel) and disease biomarkers (lower panel)
Supplementary Figure 1. Functional enrichment analyses of secretomic proteins. (a) Significant biological processes (upper panel) and disease biomarkers (lower panel) 2 involved by hrab37-mediated secretory