Fig 1: Bioactivity of recombinant SLIT1 in primary granulosa cell cultures. (A) Immunoblotting analyses of total AKT (t-AKT) and phospho-AKT (p-AKT) levels in granulosa cells treated or not with SLIT1, with ACTB (β-actin) serving as a loading control. (B) Densitometric analysis of the immunoblotting experiment depicted in A (n = 6/group). (C) Dose–response analysis of Ereg and Star mRNA levels in granulosa cells pretreated with graded doses of SLIT1, followed (or not) by LH treatment (n = 4/group). (D) Time course analysis of Ereg and Star mRNA levels in granulosa cells pretreated (or not) with SLIT1 (10 μg/ml), followed (or not) by LH treatment for the indicated times (n = 4/group). (E) Progesterone levels in spent culture media from the experiment shown in (D). Columns = means, error bars = SEM, statistically significant differences between groups (P < 0.05) are indicated with an asterisk (*) or with letters; columns lacking a common letter being significantly different from one another.
Fig 2: Recombinant SLIT1 induces apoptosis in primary granulosa cell cultures. (A) Representative photomicrographs (bar = 500 μM) of TUNEL analyses of granulosa cells pretreated (or not) with SLIT1 (10 μg/ml), followed (or) not by FSH treatment. Insets: high-magnification images (bar = 20 μM). Red = TUNEL signal, blue = DAPI counterstain. (B) Quantitative analysis of the experiment depicted in (A). (C) Immunoblotting analyses of cleaved Caspase-3 expression in granulosa cells treated or not with SLIT1 (10 μg/ml), with ACTB (β-actin) serving as a loading control. (D) Densitometric analysis of the immunoblotting experiment depicted in (C) (n = 6/group). Columns = means, error bars = SEM, statistically significant differences between groups (P < 0.001) are indicated with asterisks (***) or with letters; columns lacking a common letter being significantly different (P < 0.05) from one another.
Fig 3: Alterations in gonadotropin response gene mRNA levels in the granulosa cells of Slit1-null mice. RT-qPCR analyses of mRNA levels of the indicated genes in granulosa cells isolated from the ovaries of immature mice 48 h following eCG treatment (A), or from eCG-primed mice 4 h (B) or 12 h (C) following an ovulatory dose of hCG (n = 5/group). Columns = means, error bars = SEM, statistically significant differences between groups are indicated with asterisks (*: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0001).
Fig 4: Increased endogenous and gonadotropin-stimulated phospho-AKT levels in the granulosa cells of Slit1-null mice. (A) Immunoblotting analyses of total AKT (t-AKT) and phospho AKT (p-AKT) levels in granulosa cells from mice of the indicated genotypes, pretreated or not with SLIT1 (10 μg/ml) prior to treatment (or not) with FSH. ACTB (β-actin) served as a loading control. (B) Densitometric analysis of the immunoblotting experiment depicted in (A) (n = 6/group). (C) Same as (A), except cells were treated with LH rather than FSH. (D) Densitometric analysis of the immunoblotting experiment depicted in (C) (n = 4/group). Columns = means, error bars = SEM, statistically significant differences between genotypes (P < 0.05) are indicated with an asterisk (*); columns lacking a common letter are significantly different (P < 0.05) from one another.
Supplier Page from R&D Systems, a Bio-Techne Brand for Recombinant Mouse Slit1 Protein, CF