Fig 2: Involvement of GRP78 receptor and kinases MAP3/1 and PKA in vaspin action on P4 (A) and E2 (C) secretion as well as HSD3B (B) and CYP19A1 (D) protein expression. Representative blots from five independent experiments are shown. ELISA and Western blotting experiments were performed independently and repeated five times (n = 5). Data are plotted as the mean ± SEM. Significance between control and treatments is indicated by ***P < 0.001; or by different letters (P < 0.05), C: control.

Fig 3: (A,B) IP in murine primary Leydig cells. Total proteins collected from murine primary Leydig cell cultures were immunoprecipitated using (A) KISS1R and (B) ANKRD31 antibodies, respectively. Protein interaction among KISS1R, ANKRD31, and F-actin was detected by Western blot analysis. (C) IP in murine primary Leydig cells in vitro treated with Kp-10 alone (0.1 µM) or in combination with the specific antagonist Kp-234 (1 µM) using KISS1R antibody. Protein interaction among KISS1R, ANKRD31, and F-actin was detected by Western blot analysis. (D) Immunofluorescence analysis of F-actin, using phalloidin staining (green) in murine primary Leydig cells in vitro treated with Kp-10 alone (0.1 µM) or in combination with the specific antagonist Kp-234 (1 µM). Nuclei were labeled with TO-PRO3 iodide (blue). Scale bar: 37.5 µm. (E) Quantitative immunofluorescence analysis: F-actin signals were normalized against nuclei number, expressed in SUM(I) values and reported as mean ± SEM; experimental groups with statistically significant differences (p < 0.01) were indicated with different letters; the experimental groups without statistically significant differences were indicated with the same letter. (F) Analysis of TT content (as ng/ml) in culture media of Leydig cells in vitro treated with Kp-10 alone (0.1 µM) or in combination with the specific antagonist Kp-234 (1 µM). All the data were reported as mean ± SEM; **p < 0.01. Experimental groups with statistically significant differences (p < 0.01) were indicated with different letters. (G) Western blot analysis of CYP19 in murine primary Leydig cells in vitro treated with Kp-10 alone (0.1 µM) or in combination with the specific antagonist Kp-234 (1 µM). Signals were quantified by the densitometry analysis and normalized to Ponceau Red (Pon.S). Data are expressed in O.D. values as fold change (O.D. fc), relatively to the CTRL group, and reported as mean ± SEM; experimental groups with statistically significant differences (p < 0.01) were indicated with different letters.

Fig 4: Expression levels of P450AROM, ER-ß, Ki-67, and p53 with respect to menstrual status and tumor invasiveness. Representative immunohistochemical staining for P450AROM (a), ER-ß (b), Ki-67 (c), and p53 (d); e, g, i, and k: respective prolactinoma tissue staining scores in pre- and post-menopausal women; f, h, j, l: respective prolactinoma tissue staining scores in non-invasive and invasive prolactinomas of post-menopausal women. In a–d, magnification ×40; open arrows indicate negative immunostaining and solid arrows indicate positive immunostaining. In e–l, error bars indicate standard deviation; *P < 0.05.

Fig 5: Decreased reactive astrogliosis in GFAP-ARO-iKO mice after GCI. Western blot analyses (A) on purified astrocytes lysate isolated from sham or GCI-R3d male mouse hemisphere and densitometry analysis demonstrated significant decrease in GFAP (B) and vimentin (C) expression in the astrocytes in GFAP-ARO-iKO mice at R3d. n = 6–8 animals/group. # p < 0.05 compared to TMX (-), and * p < 0.05 compared to cre (-), one-way ANOVA followed by post hoc.