Fig. 2.

MiR-471-5p regulates germ cell apoptosis/necrosis and targets autophagy-associated proteins in Sertoli cells. a TUNEL analysis of testes sections from three miR-471-5p transgenic lines (Tg1, Tg7, and Tg13) showed significantly increased number of apoptotic/necrotic germ cells. b Electron microscopic observation of miR-471-5pTg mice testis showed drastically increased germ cell apoptosis/necrosis in two independent lines Tg1 and Tg7. Asterisks (*) indicate presence of large vacuoles in miR-471-5pTg mice. Black arrows in miR-471-5p Tg7 indicate apoptotic germ cells. WT testis showed normal acrosome and spermatogenesis. c Western blot analysis on 15p1 Sertoli cells transfected with scramble (Scr) or miR-471-5p mimic (miR-471-5p) using antibodies against Dock180 (1:1000), Becn1 (1:1000), Tecpr1 (1:1000), and Atg12 (1:1000). Gel photograph is representative of three independent experiments. Bar graphs showing quantification of band intensities are shown in Supplementary Fig. 3b. d Western blot analysis on total testis from WT and miR-471-5pTg using antibody against Dock180. β-Actin (1:50,000) was used as a loading control. Gel photograph is representative of three independent experiments (WT: n = 3 mice per experiment; miR-471-5pTg: 5 mice per experiment). Band intensities were quantified using ImageJ software, **p = 0.0016, two-tailed unpaired student t-test. e Western blot analysis on purified Sertoli cells isolated from WT and miR-471-5p Tg mice using antibodies against indicated proteins. Gel photograph is representative of three independent experiments. Purified Sertoli cells were pooled from six each of WT and miR-471-5p Tg mice/experiment. Quantification of band intensities are shown in Supplementary Fig. 3e. f–h TM4 Sertoli cells were cotransfected with scramble or miR-471-5p mimic and Renilla luciferase expression construct pRL–CMV as well as firefly luciferase construct containing Dock180 (f), ATG12 (g), or Becn1 (h) 3′-UTR. Firefly luciferase activity for each sample was normalized with Renilla luciferase activity. Graph shows mean ± SEM of three independent experiments (performed in duplicate for each experiments). ****p < 0.0001; **p = 0.0012; p = 0.0001, two-tailed unpaired student t-test. Scale bar indicates 25 μm (a), 2 μm (b)