Figure 5.

VAMP7 enhances estrogen receptor transcriptional activity. (a) Luciferase assays following transfection with VAMP7 or ESR1 or VAMP7 and ESR1 in HeLa cells incubated in absence (EtOH) or presence of 17 beta-estradiol (10⁻⁸ M) for 24 h. n = 3 independent experiments for each condition. Data are presented as means ± s.e.m. One-way analysis of variance (ANOVA) with post hoc Bonferroni test was used for statistical analyses. *P < 0.05; **P < 0.01; ***P < 0.001. (b) Western Blot analysis of ESR1 and LMNA (lamin A/C) in nuclear protein extracts of HeLa cells co-transfected with ESR1 or ESR1 and VAMP7 in absence (EtOH) or presence of 17 beta-estradiol (10⁻⁸ M) for 24 h. (c) Reciprocal co-immunoprecipitation of ESR1 and VAMP7 following their co-transfection in HeLa cells. (d) qRT-PCR analysis of key genes of ESR1 signaling in testis from WT (n = 3) and V7BAC mice (line 7; n = 3). Data are expressed as mean ± s.e.m. Mean differences between WT and V7BAC were determined by unpaired, two-tailed Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001. (e) ATF3 immunostaining of testis and external genitalia at fetal and adult stages of WT and V7BAC mice. For fetal tissues: scale bar, 250 µm. For adult urethra and testis: scale bar, 100 µm. (f) qRT-PCR of VAMP7 and ATF3 gene expression after incubation with non-targeting (scramble) or VAMP7 siRNA in NT2/D1. n = 3 independent experiments for each condition. Mean differences were determined by unpaired, two-tailed Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001. (g) Left: Heatmap of testis gene expression profiles in NT2/D1 transiently transfected with non-targeting (scramble) or VAMP7-specific siRNA. Yellow and blue colors indicate increased and decreased expression, respectively, relative to scramble. Right: Ingenuity Pathway Analysis (IPA 8.5) top canonical pathways significantly altered (20% FDR) after VAMP7 knockdown in NT2/D1 cells. (i) Pictures of inguinal hernia in V7BAC males and hematoxylin-eosin staining of extensive granulomatous inflammatory response of entrapped tissues. Scale bar, 100 µm (j) Schematic representation of VAMP7 impact on estrogen receptor signaling and male phenotypic development.