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. 2019 Dec 4;10:5530. doi: 10.1038/s41467-019-13157-7

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — Characterization of proliferation in adult cochlea. a A diagram illustrating the experimental procedure. b–f Four days post ad-Myc/ad-Cre infection of the Rosa-NICD adult (6-week-old) cochlear explant in vitro, SC mitotic figures (b, c, arrows), dividing SC nuclear doublet (b, c, arrowheads), MKI67⁺ (Ki67⁺) SCs (d, arrows) were seen. pH3⁺ SCs (e, arrows), AURBK⁺ SCs (f, arrow) and AURBK⁺ IHCs (f, arrowhead) were detected. g Alpha-tubulin labeled dividing SCs (TUBA4A⁺/ SOX2⁺/EdU⁺) in vitro, with the arrow showing TUBA4A⁺ spindle microtubules. h–k Quantification showed an increase in the SCs labeled with MKI67 (h), pH3 (i) and BrdU (j), as well as the total number of SCs (k) in ad-Myc/ad-Cre-infected compared to control ad-V5-infected Rosa-NICD cochleae. **p < 0.01, ****p < 0.0001, two-tailed unpaired (h–j) and paired (k) Student’s t-tests. Error bar, mean ± s.d.; h: n = 5; i: n = 4; j: n = 3; k: n = 9. l–n Four (l) and six (m, n) days after ad-Myc/ad-Cre injection into adult Rosa-NICD mouse cochlea in vivo, a pH3⁺ IHC (l, arrow; arrowhead points to hair bundles), a dividing IHC (m) with the arrow pointing to the cleavage site between two daughter cells, and MKI67⁺ IHCs (n, SLC17A8⁺/MKI67⁺, arrows point to a pair of IHC doublets) and SCs (n, SOX2⁺/MKI67⁺, arrowhead) were detected. o, p Compared to a control ad-V5-injected Rosa-NICD adult cochlea without proliferation (o), the ad-Myc/ad-Cre injected Rosa-NICD cochlea showed IHC proliferation with enlarged cell bodies (p, arrows show EdU⁺ IHC with enlarged bodies), 12 days after injection. q, r Quantification showed increase in MKI67⁺ IHCs (q) and pH3⁺ IHCs (r) in adult Rosa-NICD cochleae injected with ad-Myc/ad-Cre or ad-V5 for 4 days, respectively. **p < 0.01, two-tailed unpaired Student’s t-test. Error bar, mean ± s.d.; n = 8 for each group in (q); n = 6 for each group in (r). s, t Quantification showed increase in BrdU⁺ IHCs (s) and the total number of IHCs (t) in adult Rosa-NICD cochleae injected with ad-Myc/ad-Cre and ad-V5 in vivo at six days. ****p < 0.0001, two-tailed unpaired Student’s t-test. Error bar, mean ± s.d.; n = 8 for each group in (s, t). n is the number of biologically independent samples. Scale bars: 10 μm. Source data are provided as a Source Data file.