ROS generation and enhanced proliferation of GS cells by Nox1 transfection. ROS generation and enhanced proliferation of GS cells by Nox1 transfection.

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ROS generation and enhanced proliferation of GS cells by Nox1 transfection. ROS generation and enhanced proliferation of GS cells by Nox1 transfection. (A) Real-time PCR analysis of Nox1-related components in GS cells (n = 3 cultures). (B) Real-time PCR analysis of Nox1-related components by FGF2 and GDNF (FG) supplementation (n = 3 cultures). The cells were starved for 3 d, and FGF2 and GDNF were added for 6 h before sample collection. *P < 0.05 (t test). (C) Real-time PCR analysis of Nox1-related components 2 (DPI) or 3 d (SB203589 and XMD 8-92) after supplementation with the indicated inhibitors (n = 3 cultures). Mean values for control experiment are indicated by the horizontal line. *P < 0.05 (t test). (D) Real-time PCR analysis of indicated genes 10 min after hydrogen peroxide (90 μM) supplementation (n = 3 cultures). *P < 0.05 (t test). (E) Flow cytometric analysis of ROS generation 3 h after Nox1 transfection (n = 3 cultures; moi = 10). (F) Cell recovery 5 d after Nox1 or Noxa2 transfection (n = 6 cultures; moi = 10). *P < 0.05 (ANOVA). (G) Flow cytometric analysis of ROS generation 6 d after Noxa2 depletion (n = 3 cultures; moi = 4). *P < 0.05 (linear regression). (H) Cell recovery 7 d after Noxa2 depletion (n = 6 cultures; moi = 4). *P < 0.05 (t test). (I) Flow cytometric analysis of ROS generation 3 h after Noxa2 transfection (n = 3 cultures; moi = 4). (J) Flow cytometric analysis of ROS generation 3 h after Nox1 transfection into GS cells, in which Noxa2 was depleted 6 d before (n = 3 cultures; moi = 4). Eyfp was transfected into Noxa2-depleted cells as a control. (K) Colony counts after spermatogonial transplantation of GS cells transfected with the indicated genes (n = 12 testes). *P < 0.05 (t test). Data information: in (A–K), data are presented as mean ± SEM. Hiroko Morimoto et al. LSA 2019;2:e201900374 © 2019 Morimoto et al.