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. 2021 Sep 13;11(19):9397–9414. doi: 10.7150/thno.57037

Figure 2.

Figure 2

Snhg1 overexpression promoted P7 CM proliferation. (A) EdU staining of P7 CMs transduced with Adv-NC or Adv-Snhg1, (647 CMs from 5 mice in the Adv-NC group, 723 CMs from 5 mice in the Adv-Snhg1group). CMs were immunostained for cardiac troponin T (cTnT) and nuclei were stained with DAPI. Arrows indicate positive CMs. (B) Immunostaining for pH3 of P7 CMs transduced with Adv-NC or Adv-Snhg1, (467 CMs from 5 mice in the Adv-NC group, 539 CMs from 5 mice in the Ad-Snhg1 group). (C-D) Immunostaining for Aurora B of P7 CMs transduced with Adv-NC or Adv-Snhg1, (536 CMs from 5 mice in the Adv-NC group, 652 CMs from 5 mice in the Adv-Snhg1 group). (E) Detection of cell cycle alterations in P7 CMs transfected with Adv-Snhg1 or Adv-NC by flow cytometry. (F) Time-lapse images of Snhg1-induced cytokinesis in P7 mouse CMs isolated from MYH6-mCherry transgenic mice (427 CMs from 5 mice in the Adv-Snhg1group). Panels are representative of images recorded during cell division (see Video 1). Arrows indicate CMs undergoing cell division. (G) Quantification of CM binucleation in P7 CMs transduced with Adv-Snhg1 or Adv-NC. 428 CMs from 5 mice in Adv-Snhg1 group at 24 h, 476 CMs from 5 mice in Adv-NC group at 48 h, 567 CMs from 5 mice in Adv-Snhg1 group at 24 h, 536 CMs from 5 mice in Adv-NC group at 48 h. Nucleation status of EdU+ CMs was assessed by immunostaining for cardiac troponin T (cTnT), EdU and N-cadherin (N-cad). (H) The binucleation index, defined as the relative increase in the percentage of binuclear cells, within the EdU+ CM population from 24 to 48 h. Calculated from (G). (I) Schematic of the experiments in day 7 mouse hearts injected with AAV9 vectors. IF, immunofluorescence. (J-K) Immunostaining for Ki67 in P7 mouse hearts (n = 5). Arrows indicate positive CMs. (L) Immunostaining for pH3 in P7 mouse hearts (n = 5). Arrows indicate positive CMs. (M) EdU incorporation was detected in CM nuclei (376 CMs from 5 mice in the AAV9-NC group, 413 CMs from 5 mice in the AAV9-Snhg1 group). (N-O) Immunostaining for Ki67 and Aurora B in P7 MYH6-mCherry transgenic mice (n = 5). All CMs were immunostained for cTnT and nuclei were stained with DAPI. Arrows indicate positive CMs.