Journal: Cell Communication and Signaling : CCS

Article Title: Slit/Robo signaling regulates Leydig cell steroidogenesis

doi: 10.1186/s12964-020-00696-6

Figure Lengend Snippet: Quantitative RT-qPCR primer sequences

Article Snippet: The next day, the culture medium was replaced with a serum-free medium, and the cells incubated overnight before treatment with vehicle (PBS) or SLIT recombinant mouse protein (R&D Systems, Minneapolis, MN, USA, SLIT1 #5199-SL, SLIT2 #5444-SL, SLIT3 #9295-SL) at varying concentrations and for varying times.

Techniques:

Journal: Cell Communication and Signaling : CCS

Article Title: Slit/Robo signaling regulates Leydig cell steroidogenesis

doi: 10.1186/s12964-020-00696-6

Figure Lengend Snippet: Antibodies

Article Snippet: The next day, the culture medium was replaced with a serum-free medium, and the cells incubated overnight before treatment with vehicle (PBS) or SLIT recombinant mouse protein (R&D Systems, Minneapolis, MN, USA, SLIT1 #5199-SL, SLIT2 #5444-SL, SLIT3 #9295-SL) at varying concentrations and for varying times.

Techniques: Western Blot, Immunohistochemistry

Journal: Cell Communication and Signaling : CCS

Article Title: Slit/Robo signaling regulates Leydig cell steroidogenesis

doi: 10.1186/s12964-020-00696-6

Figure Lengend Snippet: Slit and Robo genes are expressed in the Leydig cells of the mouse testis. a RT-qPCR analyses of brain, testicular and purified Leydig cell Slit and Robo mRNA levels (n = 4–6 per group). The relative levels are represented as delta-Ct versus Actb . Data are means ± sem. b SLIT1, -2 and -3 and ROBO1 immunohistochemistry analyses of 8 week-old wild-type mouse testis

Article Snippet: The next day, the culture medium was replaced with a serum-free medium, and the cells incubated overnight before treatment with vehicle (PBS) or SLIT recombinant mouse protein (R&D Systems, Minneapolis, MN, USA, SLIT1 #5199-SL, SLIT2 #5444-SL, SLIT3 #9295-SL) at varying concentrations and for varying times.

Techniques: Quantitative RT-PCR, Purification, Immunohistochemistry

Journal: Cell Communication and Signaling : CCS

Article Title: Slit/Robo signaling regulates Leydig cell steroidogenesis

doi: 10.1186/s12964-020-00696-6

Figure Lengend Snippet: Exogenous SLIT2 decreases steroidogenesis in Leydig cells in vitro. Expression of Star , Cyp11a1 and Cyp17a1 determined by RT-qPCR in MA10 cells treated a for 4 h with vehicle or 1, 5 and 10 µg/ml exogenous SLIT2; b for 2, 4, 8 and 24 h with vehicle or 10 µg/ml SLIT2. c Representative graph of progesterone concentrations (corrected to RNA input) measured in the spent culture media of MA10 cells treated for 8 h with vehicle or 10 µg/ml SLIT2. d Expression of Star , Cyp11a1 and Cyp17a1 determined by RT-qPCR and e representative graph of testosterone concentrations (corrected to RNA input) measured in the spent culture media of mouse primary Leydig cell cultures treated for 8 h with vehicle or 10 µg/ml SLIT2. Experiments were performed three times in triplicate. Expression of each transcript was normalized to the housekeeping gene Rplp0 . Data are means ± sem; statistical analysis (Student’s T -test): * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: The next day, the culture medium was replaced with a serum-free medium, and the cells incubated overnight before treatment with vehicle (PBS) or SLIT recombinant mouse protein (R&D Systems, Minneapolis, MN, USA, SLIT1 #5199-SL, SLIT2 #5444-SL, SLIT3 #9295-SL) at varying concentrations and for varying times.

Techniques: In Vitro, Expressing, Quantitative RT-PCR

Journal: Cell Communication and Signaling : CCS

Article Title: Slit/Robo signaling regulates Leydig cell steroidogenesis

doi: 10.1186/s12964-020-00696-6

Figure Lengend Snippet: Exogenous SLIT2 decreases CREB phosphorylation. a Quantification of total and phosphorylated CREB, AKT and mTOR protein levels normalized to ACTB in MA10 cells treated for 1 h with vehicle or 10 µg/ml SLIT2. Quantification of total and phosphorylated AKT and CREB protein levels normalized to GAPDH, and expression of Star determined by RT-qPCR, in MA10 cells treated with vehicle or b 100 nM wortmannin, or c 20 µg/ml SC79, for 1 h (protein) or 4 h (mRNA). n = 3–9 samples per group. Expression of Star was normalized to the housekeeping gene Rplp0 . Data are means ± sem; statistical analysis (Student’s T -test): * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: The next day, the culture medium was replaced with a serum-free medium, and the cells incubated overnight before treatment with vehicle (PBS) or SLIT recombinant mouse protein (R&D Systems, Minneapolis, MN, USA, SLIT1 #5199-SL, SLIT2 #5444-SL, SLIT3 #9295-SL) at varying concentrations and for varying times.

Techniques: Expressing, Quantitative RT-PCR

Journal: Cell Communication and Signaling : CCS

Article Title: Slit/Robo signaling regulates Leydig cell steroidogenesis

doi: 10.1186/s12964-020-00696-6

Figure Lengend Snippet: Exogenous SLIT2 alters Leydig cell responsiveness to LH by decreasing Lhcgr expression. a Expression of Lhcgr determined by RT-qPCR in MA10 cells treated for 2, 4, 8 and 24 h with vehicle or 10 µg/ml SLIT2. b Expression of Star , Cyp11a1 , Cyp17a1 and Hsd3b1 determined by RT-qPCR in MA10 cells treated with 10 µg/ml SLIT2 for 24 h, ± 50 ng/ml LH for 4 h. c Quantification of total and phospho-CREB protein levels normalized to GAPDH in MA10 cells treated with 10 µg/ml SLIT2 for 24 h, ± 50 ng/ml LH for 30 min. d Expression of Star and Cyp11a1 determined by RT-qPCR in MA10 cells treated with 10 µg/ml SLIT2 for 24 h, ± 10 µM forskolin for 4 h. n = 3–9 samples per group. Expression of each transcript was normalized to the housekeeping gene Rplp0 . Data are means ± sem; statistical analysis (Student’s T -test): * p < 0.05; ** p < 0.01; *** p < 0.001; # p < 0.05; ## p < 0.01; ### p < 0.001; NS not significant

Article Snippet: The next day, the culture medium was replaced with a serum-free medium, and the cells incubated overnight before treatment with vehicle (PBS) or SLIT recombinant mouse protein (R&D Systems, Minneapolis, MN, USA, SLIT1 #5199-SL, SLIT2 #5444-SL, SLIT3 #9295-SL) at varying concentrations and for varying times.

Techniques: Expressing, Quantitative RT-PCR

Journal: Cell Communication and Signaling : CCS

Article Title: Slit/Robo signaling regulates Leydig cell steroidogenesis

doi: 10.1186/s12964-020-00696-6

Figure Lengend Snippet: Increased testicular steroidogenesis in Robo1- null mice. a Expression of Star , Cyp11a1 and Cyp17a1 determined by RT-qPCR in primary Leydig cells isolated from Robo1 +/+ vs Robo1 Robo1 −/− mice treated for 8 h with vehicle or 10 µg/ml SLIT2. n = 4 samples per group. b Expression of Robo2 determined by RT-qPCR in cultured Leydig cells isolated from Robo1 +/+ and Robo 1 −/− mice. n = 4 samples per group. c Expression of Star , Cyp11a1 , Cyp17a1 , Hsd3b1 and Lhcgr determined by RT-qPCR in testes from four month-old Robo1 +/+ and Robo1 −/− mice. n = 5–7 per group. Expression of each transcript was normalized to the housekeeping gene Rplp0 . d Intra-testicular testosterone concentrations corrected to testis weight measured in two- and four month-old Robo1 +/+ and Robo1 −/− mice, and serum LH levels measured in four month-old mice. n = 5–10 per group. Data are means ± sem; statistical analysis (Student’s T -test): * p < 0.05; ** p < 0.01; *** p < 0.001; # p < 0.05; ## p < 0.01; ### p < 0.001

Article Snippet: The next day, the culture medium was replaced with a serum-free medium, and the cells incubated overnight before treatment with vehicle (PBS) or SLIT recombinant mouse protein (R&D Systems, Minneapolis, MN, USA, SLIT1 #5199-SL, SLIT2 #5444-SL, SLIT3 #9295-SL) at varying concentrations and for varying times.

Techniques: Expressing, Quantitative RT-PCR, Isolation, Cell Culture

Journal: Cell

Article Title: Evolution of Cortical Neurogenesis in Amniotes Controlled by Robo Signaling Levels

doi: 10.1016/j.cell.2018.06.007

Figure Lengend Snippet: Functional Validation of Genetic Reagents and Test of Interaction between Robo and Notch Signaling, Related to , , and (A and B) Validation of dnRobo and myrRobo as dominant-negative and constitutively active for Robo signaling, respectively. In (A), growth cone collapse assay of growing axons from explants of embryonic mouse retinas, electroporated to express Gfp or dnRobo and exposed to recombinant Slit protein or vehicle solution. Failure of response to Slit upon dnRobo-overexpression demonstrates its dominant-negative effect (n = 44-58 growth cones per group, 3 independent experiments). In (B), branching assay of growing axons from single neurons of embryonic rat dorsal root ganglion, overexpressing Gfp alone or with myrRobo constructs as indicated. Exuberant axonal branching typically elicited by Slit-Robo signaling occurs in myrRobo-expressing neurons in the absence of Slit, demonstrating constitutive activation of Robo signaling (n = 5-10 neurons per group). (C–F) Validation of crispr constructs for disruption of Dll1 in mouse and human. (C) Top, sequence of the gRNA targeting mouse Dll1 , and schematic of the orientation and location of the targeting site (black arrow) within the mDll1 coding sequence (gray bar). Bottom, validation of Crispr-mediated editing of the mDll1 locus upon electroporation with gDll1 plus Cas9, but not with Cas9 alone. Different lanes correspond to independent electroporated embryos. M, molecular weight marker. Arrow indicates 1,025 bp amplicon, arrowheads indicate the products of PCR amplicon digestion by Syrveyor Nuclease (656 + 368 bp), absent in the Cas9-alone lanes. (D) Left, sequence of the gRNA targeting human Dll1 , and schematic of the orientation and location of the targeting site (black arrow) within the hDll1 coding sequence (gray bar). Right, chromograms for genome sequence validation of Crispr-mediated editing of the hDll1 locus upon electroporation of cerebral organoid with gDll1 plus Cas9. A 270bp fragment was inserted at position 54 of the coding sequence, introducing a STOP codon in position 76. (E and F) Effect of electroporating crDll1 in NCx (green cells) on the abundance of Dll1 protein (red). Details are examples VZ cells loosing Dll1 protein (arrowheads) from the cell surface upon crDll1 (n = 3 embryos per group). (G–K) Antibody stain for GFP and Robo1 or Dll1 in NCx at E13.5 upon electroporation of the indicated plasmid combinations at E12.5, and quantifications (paired t test). Arrowheads indicate area of increased Robo (n = 3 embryos per group). Values are mean + SEM; paired or independent samples t tests; ∗ p < 0.05; ns, not significant. Scale bars: 30 μm (E), 50 μm (G and H).

Article Snippet: After 24h of incubation, recombinant mouse Slit protein (R&D Systems) was added to the medium (250ng/μl) and the explants were fixed 1h later with PFA 2% during 15 min.

Techniques: Functional Assay, Biomarker Discovery, Dominant Negative Mutation, Recombinant, Over Expression, Construct, Expressing, Activation Assay, CRISPR, Disruption, Sequencing, Electroporation, Molecular Weight, Marker, Amplification, Staining, Plasmid Preparation

Journal: Current biology : CB

Article Title: FLRT3 is a Robo1-interacting protein that determines Netrin-1 attraction in developing axons.

doi: 10.1016/j.cub.2014.01.042

Figure Lengend Snippet: Figure 2. Slit1 Enables Netrin-1 Attraction by Activating the Robo1 Receptor

Article Snippet: Recombinant Slit1 (5 nM; R&D Systems), FLRT3-ECD [15] (1 mg/ml), a mouse anti-DCC antibody (AF5 clone; Abcam), or control mouse IgG was (J and K) Robo1+/+ rTCAs grow rostrally as a compact bundle (J), whereas the (L) Quantification of the data represented in (J) and (K). rTCA dispersion at the v tailed Student’s t test. (M) Scheme representing the topographic guidance of rTCAs and iTCAs in t Slit1 and Netrin-1 [21].

Techniques: