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slit3 recombinant protein (R&D Systems)

Name: slit3 recombinant protein
Brand: R&D Systems
Rating: 93 (3 reviews)

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R&D Systems slit3 recombinant protein
Slit3 Recombinant Protein, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/slit3 recombinant protein/product/R&D Systems
Average 93 stars, based on 3 article reviews

slit3 recombinant protein - by Bioz Stars, 2026-02

93/100 stars

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ROBO2 is overexpressed in all four TNBC cell lines when these cells are incubated with SLIT3. Hence, the ectopic SLIT3 reprograms cancer cells. Each condition had six replicates with at least two fields of view per well imaged. ** p -value < 0.001.

Journal: International Journal of Molecular Sciences

Article Title: CD36 + Fibroblasts Secrete Protein Ligands That Growth-Suppress Triple-Negative Breast Cancer Cells While Elevating Adipogenic Markers for a Model of Cancer-Associated Fibroblast

doi: 10.3390/ijms232112744

Figure Lengend Snippet: ROBO2 is overexpressed in all four TNBC cell lines when these cells are incubated with SLIT3. Hence, the ectopic SLIT3 reprograms cancer cells. Each condition had six replicates with at least two fields of view per well imaged. ** p -value < 0.001.

Article Snippet: Human recombinant proteins, SLIT3 (Novus Biological 9255-SL-050 (Littleton, CO, USA)), FBLN1 (Abbexa abx066632), and PENK (Abbexa abx650333), were commercially acquired. (MET5) Enkephalin (Sigma M6638 ((St. Louis, MI, USA))) was also purchased commercially.

Techniques: Incubation

Robo4-Ig inhibits Slit3-induced angiogenesis. ( A ). Robo4-Ig bound Slit3. Robo4-Ig was immobilized in an ELISA plate and incubated with His-tagged Slit3. The bound Slit3 was measured by ELISA using an HRP-conjugated anti-His antibody. BSA was used as a background control and for normalization. ( B ). Robo4-Ig inhibited Slit3 binding to dEC surface. Confluent dECs were fixed and incubated with His-tagged Slit3 or BSA in the absence or presence of Robo4-Ig. The cell surface-bound Slit3 was quantified by ELISA using an HRP-conjugated anti-His antibody. BSA was used as a background control and for normalization. ( C , D ) Robo4-Ig inhibited Slit3-induced dEC proliferation and migration. Slit3-induced dEC proliferation in DMEM containing 0.2% serum (36-h, C ) and Slit3-induced transwell cell migration in serum-free DMEM (9-h, D ) were determined in the absence or presence of Robo4-Ig. BSA was included as a background control. ( E – G ) Robo4-Ig inhibited Slit3-induced neovascularization in matrigel plug assay. The Slit3- or BSA-supplemented growth factor reduced matrigel with or without Robo4-Ig was implanted s.c. into adult female mice, and the plugs were harvested two weeks later. Representative gross images ( E ) and neo-vasculature staining ( F ) are shown , including CD31 staining (green) of endothelial cells, DAPI staining (blue) of cellularity ( F ), and hemoglobin content in the Matrigel plugs ( G ), N = 6 per group. ( H ). Anti-Unc5B neutralizing antibody treatment in the VEGF165—or Slit3-induced dEC migration. VEGF165- (100 ng/ml) or Slit3-induced dEC transwell cell migration in serum-free DMEM was determined in the presence of native IgG or anti-Unc5B neutralizing IgG antibody (2 μg/ml). The data shown in A-D and H represent 3 independent experiments. All the data are presented as mean ± SD. The student's t-test was performed for a two-group comparison and ANOVA for multiple groups. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Journal: Scientific Reports

Article Title: Robo4 is constitutively shed by ADAMs from endothelial cells and the shed Robo4 functions to inhibit Slit3-induced angiogenesis

doi: 10.1038/s41598-022-08227-8

Figure Lengend Snippet: Robo4-Ig inhibits Slit3-induced angiogenesis. ( A ). Robo4-Ig bound Slit3. Robo4-Ig was immobilized in an ELISA plate and incubated with His-tagged Slit3. The bound Slit3 was measured by ELISA using an HRP-conjugated anti-His antibody. BSA was used as a background control and for normalization. ( B ). Robo4-Ig inhibited Slit3 binding to dEC surface. Confluent dECs were fixed and incubated with His-tagged Slit3 or BSA in the absence or presence of Robo4-Ig. The cell surface-bound Slit3 was quantified by ELISA using an HRP-conjugated anti-His antibody. BSA was used as a background control and for normalization. ( C , D ) Robo4-Ig inhibited Slit3-induced dEC proliferation and migration. Slit3-induced dEC proliferation in DMEM containing 0.2% serum (36-h, C ) and Slit3-induced transwell cell migration in serum-free DMEM (9-h, D ) were determined in the absence or presence of Robo4-Ig. BSA was included as a background control. ( E – G ) Robo4-Ig inhibited Slit3-induced neovascularization in matrigel plug assay. The Slit3- or BSA-supplemented growth factor reduced matrigel with or without Robo4-Ig was implanted s.c. into adult female mice, and the plugs were harvested two weeks later. Representative gross images ( E ) and neo-vasculature staining ( F ) are shown , including CD31 staining (green) of endothelial cells, DAPI staining (blue) of cellularity ( F ), and hemoglobin content in the Matrigel plugs ( G ), N = 6 per group. ( H ). Anti-Unc5B neutralizing antibody treatment in the VEGF165—or Slit3-induced dEC migration. VEGF165- (100 ng/ml) or Slit3-induced dEC transwell cell migration in serum-free DMEM was determined in the presence of native IgG or anti-Unc5B neutralizing IgG antibody (2 μg/ml). The data shown in A-D and H represent 3 independent experiments. All the data are presented as mean ± SD. The student's t-test was performed for a two-group comparison and ANOVA for multiple groups. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: His-tagged recombinant mouse Slit3 (3629-SL-050) was from R&D Systems, PMA (ab120297) from Abcam, TAPI-2 (187034-31-7) from Cayman Chemical, GW282026X (AOB3632) from AOBIOUS Inc., Polybrene (sc-134220) from Santa Cruz Biotechnology, and GI254023x (260264-93-5), LPS (#L7770) and PEI (#764965) were from Sigma-Aldrich.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Control, Binding Assay, Migration, Matrigel Assay, Staining, Comparison

Slit3 down-regulates cell surface Robo4 and induces Robo4 internalization. ( A ) GW but not Slit3 reduced Cdh5 shedding. dECs in serum-free cultured were supplemented with BSA or Slit3 with or without GW, the duo-inhibitor for ADAM10 and ADAM. The conditioned media and cell lysate were probed with an anti-Cdh5 antibody in Western blot. ( B ) Slit3 reduced cell surface Robo4, and this was blocked by EIPA. Starved dECs were treated with Slit3 or BSA in the absence or presence of EIPA, and then cell surface Robo4 was assessed by flow cytometry after staining with an anti-Robo4 ectodomain antibody. Anti-Robo4 IgG and naïve IgG staining are drawn in heavy-bright and thin-faint lines, respectively, with corresponding colors. ( C ) Slit3 induced cell surface Robo4 internalization, and this was blocked by EIPA. After biotinylation of cell surface proteins at 4 °C, the hRobo4-HA-FLAG expressing dECs were treated with Slit3 or BSA in the absence or presence of EIPA at 37 °C. Cells were lysed immediately (total biotinylated protein preserved) or after MENSA treatment (MESNA cleaves uninternalized biotinylated protein), and biotinylated proteins were pulled down using streptavidin-coated beads. hRobo4-HA-FLAG were probed in western blot with anti-FLAG antibody. ( D ) Slit3 inhibited Robo4 shedding, and this was restored by EIPA. dECs were treated with Slit3 or BSA in the absence or presence of EIPA, and then sRobo4 in conditioned medium, and total Robo4 and actin in cell lysates were probed. sRobo4 was normalized to the total Robo4. The data represent 3 independent experiments and are presented as mean ± SD. The student's t-test was performed for a two-group comparison. ns, not significant; *p < 0.05; **p < 0.01.

Journal: Scientific Reports

Article Title: Robo4 is constitutively shed by ADAMs from endothelial cells and the shed Robo4 functions to inhibit Slit3-induced angiogenesis

doi: 10.1038/s41598-022-08227-8

Figure Lengend Snippet: Slit3 down-regulates cell surface Robo4 and induces Robo4 internalization. ( A ) GW but not Slit3 reduced Cdh5 shedding. dECs in serum-free cultured were supplemented with BSA or Slit3 with or without GW, the duo-inhibitor for ADAM10 and ADAM. The conditioned media and cell lysate were probed with an anti-Cdh5 antibody in Western blot. ( B ) Slit3 reduced cell surface Robo4, and this was blocked by EIPA. Starved dECs were treated with Slit3 or BSA in the absence or presence of EIPA, and then cell surface Robo4 was assessed by flow cytometry after staining with an anti-Robo4 ectodomain antibody. Anti-Robo4 IgG and naïve IgG staining are drawn in heavy-bright and thin-faint lines, respectively, with corresponding colors. ( C ) Slit3 induced cell surface Robo4 internalization, and this was blocked by EIPA. After biotinylation of cell surface proteins at 4 °C, the hRobo4-HA-FLAG expressing dECs were treated with Slit3 or BSA in the absence or presence of EIPA at 37 °C. Cells were lysed immediately (total biotinylated protein preserved) or after MENSA treatment (MESNA cleaves uninternalized biotinylated protein), and biotinylated proteins were pulled down using streptavidin-coated beads. hRobo4-HA-FLAG were probed in western blot with anti-FLAG antibody. ( D ) Slit3 inhibited Robo4 shedding, and this was restored by EIPA. dECs were treated with Slit3 or BSA in the absence or presence of EIPA, and then sRobo4 in conditioned medium, and total Robo4 and actin in cell lysates were probed. sRobo4 was normalized to the total Robo4. The data represent 3 independent experiments and are presented as mean ± SD. The student's t-test was performed for a two-group comparison. ns, not significant; *p < 0.05; **p < 0.01.

Techniques: Cell Culture, Western Blot, Flow Cytometry, Staining, Expressing, Comparison

sRobo4 generation and its role in angiogenic Slit3-Robo4 signaling. Under the unstimulated condition, the Robo4 ectodomain is constitutively cleaved by ADAM10 and ADAM17 to generate sRobo4. The generated sRobo4 blocks Slit3-Robo4 interaction, thereby inhibiting angiogenic Slit3 signaling. Meanwhile, Slit3 inhibits Robo4 shedding by inducing Robo4 internalization to shield the receptor from shedding.

Journal: Scientific Reports

Article Title: Robo4 is constitutively shed by ADAMs from endothelial cells and the shed Robo4 functions to inhibit Slit3-induced angiogenesis

doi: 10.1038/s41598-022-08227-8

Figure Lengend Snippet: sRobo4 generation and its role in angiogenic Slit3-Robo4 signaling. Under the unstimulated condition, the Robo4 ectodomain is constitutively cleaved by ADAM10 and ADAM17 to generate sRobo4. The generated sRobo4 blocks Slit3-Robo4 interaction, thereby inhibiting angiogenic Slit3 signaling. Meanwhile, Slit3 inhibits Robo4 shedding by inducing Robo4 internalization to shield the receptor from shedding.

Techniques: Generated

Figure 1. SLIT3 is present at high levels in fibrillar collagen–producing cells. (A) Linear regression analysis between the transcript levels of SLIT3 and COL1A1, SLIT3 and COL3A1, and ROBO1 and COL1A1 across 512 cell lines from the FANTOM5 project (28). TPM, transcripts per million. (B) Single-cell transcriptome data from the Tabula Muris project (29), with t-distributed stochastic neighbor embedding plot of all cells collected by FACS, overlaid with the pre- dominant cell type composing each cluster (n = 44,949 individual cells from 20 mouse organs). The clusters of cells expressing Slit3, Col1a1, and Robo1. (C) Transcript levels of Slit3, Col1a1, and Robo1 in cardiac fibroblasts, left ventricle, freshly isolated cardiomyocytes, aortic adventitial fibroblasts, lung fibroblasts, aortic vascular smooth muscle cells (VSMC), aorta media, and aorta adventitia from WT mice. Samples taken directly from or isolated from living tissue are marked blue and samples of purified and cultured cells are marked red on panels. Each data point represents tissue/cells obtained from a single animal. (D) Confocal immunofluorescence images of mouse aortic adventitial fibroblasts stained with SLIT3 (shown in green), ROBO1 (shown in green), and DAPI (shown in blue) (n = 2). Scale bars: 50 μm. *P < 0.05, **P < 0.01, and ***P < 0.001 using 1-way ANOVA with Tamhane T2 multiple comparisons test (C).

Journal: JCI insight

Article Title: SLIT3 deficiency attenuates pressure overload-induced cardiac fibrosis and remodeling.

doi: 10.1172/jci.insight.136852

Figure Lengend Snippet: Figure 1. SLIT3 is present at high levels in fibrillar collagen–producing cells. (A) Linear regression analysis between the transcript levels of SLIT3 and COL1A1, SLIT3 and COL3A1, and ROBO1 and COL1A1 across 512 cell lines from the FANTOM5 project (28). TPM, transcripts per million. (B) Single-cell transcriptome data from the Tabula Muris project (29), with t-distributed stochastic neighbor embedding plot of all cells collected by FACS, overlaid with the pre- dominant cell type composing each cluster (n = 44,949 individual cells from 20 mouse organs). The clusters of cells expressing Slit3, Col1a1, and Robo1. (C) Transcript levels of Slit3, Col1a1, and Robo1 in cardiac fibroblasts, left ventricle, freshly isolated cardiomyocytes, aortic adventitial fibroblasts, lung fibroblasts, aortic vascular smooth muscle cells (VSMC), aorta media, and aorta adventitia from WT mice. Samples taken directly from or isolated from living tissue are marked blue and samples of purified and cultured cells are marked red on panels. Each data point represents tissue/cells obtained from a single animal. (D) Confocal immunofluorescence images of mouse aortic adventitial fibroblasts stained with SLIT3 (shown in green), ROBO1 (shown in green), and DAPI (shown in blue) (n = 2). Scale bars: 50 μm. *P < 0.05, **P < 0.01, and ***P < 0.001 using 1-way ANOVA with Tamhane T2 multiple comparisons test (C).

Article Snippet: Human TGF-β1 (240-B, R&D Systems, Bio-Techne), rat PDGF-BB (520-BB/CF, R&D Systems, Bio-Techne), human FGF-basic (03-0002, STEMGENT), and mouse Slit3 (aa 34-1116, 9296-SL-050, R&D Systems, Bio-Techne) were used at the indicated concentrations.

Techniques: Expressing, Isolation, Purification, Cell Culture, Immunofluorescence, Staining

Figure 2. SLIT3 deficiency reduces fibrillar collagen production in vivo. (A) Masson’s trichrome–stained histological sections of lung, spleen, and kidney in 8-week-old Slit3−/− and WT mice. Results are representative of samples obtained from 5 animals per genotype group. Scale bars: 100 μm, 60 μm, and 100 μm from top to bottom. (B) Transcript levels of Slit3, Col1a1, and Col3a1 in the aortic adventitia, lung, spleen, and kidney in 8-week-old Slit3−/− and WT mice (n = 4–6 per group). (C) Linear regression analysis between the transcript levels of Slit3 and Col1a1 in the aortic adventitia, lung, spleen, and kidney from 8-week-old WT mice (n = 5–7 per group). (D) Quantification of tissue collagen content by assessment of hydroxyproline concentrations in the femur, skin, lung, spleen, and kidney in 8-week-old Slit3−/− and WT mice (n = 5–8 per group). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. WT mice using the unpaired 2-tailed Student’s t test (B and D). n, number; M, male; F, female.

Journal: JCI insight

Article Title: SLIT3 deficiency attenuates pressure overload-induced cardiac fibrosis and remodeling.

doi: 10.1172/jci.insight.136852

Figure Lengend Snippet: Figure 2. SLIT3 deficiency reduces fibrillar collagen production in vivo. (A) Masson’s trichrome–stained histological sections of lung, spleen, and kidney in 8-week-old Slit3−/− and WT mice. Results are representative of samples obtained from 5 animals per genotype group. Scale bars: 100 μm, 60 μm, and 100 μm from top to bottom. (B) Transcript levels of Slit3, Col1a1, and Col3a1 in the aortic adventitia, lung, spleen, and kidney in 8-week-old Slit3−/− and WT mice (n = 4–6 per group). (C) Linear regression analysis between the transcript levels of Slit3 and Col1a1 in the aortic adventitia, lung, spleen, and kidney from 8-week-old WT mice (n = 5–7 per group). (D) Quantification of tissue collagen content by assessment of hydroxyproline concentrations in the femur, skin, lung, spleen, and kidney in 8-week-old Slit3−/− and WT mice (n = 5–8 per group). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. WT mice using the unpaired 2-tailed Student’s t test (B and D). n, number; M, male; F, female.

Techniques: In Vivo, Staining

Figure 3. SLIT3 deficiency reduces cardiac fibrillar collagen production and LV biomechanical toughness. (A) Representative Masson’s trichrome–stained histological sections of heart in 8-week-old and 1-year-old Slit3−/− and WT mice; representative confocal immunofluorescence images of heart stained with collagen type I (red), CD31 (green), and DAPI (blue) in 8-week-old Slit3−/− and WT mice; and representative immunohistochemical stain for CD31 in sections of hearts from 8-week-old Slit3−/− and WT mice (n = 3 per group). (B) Transcript levels of Slit3, Col1a1, and Col3a1 in the left ventricle and right ventricle in 8-week-old Slit3−/− and WT mice (n = 4–6 per group). (C) Linear regression analysis between the transcript levels of Slit3 and Col1a1 in the left ventricle and right ventricle from 8-week-old WT mice (n = 6 per group). (D) Transcript levels of Slit3 in the left and right ventricle, and their linear regression analysis in 8-week-old WT mice (n = 6 per group). (E) Quantification of perivascular collagen area of coronary arteries and mitral ratio of peak early to late diastolic filling velocity (mitral E/A ratio) in 8-week-old and 1-year-old Slit3−/− and WT mice (n = 4–16 per group). (F) Quantification of cardiac collagen content by assessment of hydroxyproline concentrations in 8-week-old Slit3−/− and WT mice (female, n = 8 per group). (G) Representative passive LV pressure-volume curve and quantification of energy density required to rupture the cardioplegia-relaxed left ventricles by balloon catheter inflation in 8-week-old Slit3−/− and WT mice (n = 4 per group). (H) Representative image of myocardial rupture and overall survival curve of the first week after left anterior descending coronary artery ligation (myocardial infarction, MI) in 8-week-old Slit3−/− and WT mice. The blue arrowhead indicates the position of the myocardial rupture hole, and survival analysis was performed using the Kaplan-Meier method. Log-rank test, P = 0.0039 (n = 24–25 per group). (I) Representative image of femurs or hearts, as well as quan- tification of femur or heart weight/tibia length ratio in 8-week-old Slit3−/− and WT mice (n = 6 per group). Scale bar: 1 mm. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. WT mice using the unpaired 2-tailed Student’s t test (B, E–G, and I). n, number; M, male; F, female.

Journal: JCI insight

Article Title: SLIT3 deficiency attenuates pressure overload-induced cardiac fibrosis and remodeling.

doi: 10.1172/jci.insight.136852

Figure Lengend Snippet: Figure 3. SLIT3 deficiency reduces cardiac fibrillar collagen production and LV biomechanical toughness. (A) Representative Masson’s trichrome–stained histological sections of heart in 8-week-old and 1-year-old Slit3−/− and WT mice; representative confocal immunofluorescence images of heart stained with collagen type I (red), CD31 (green), and DAPI (blue) in 8-week-old Slit3−/− and WT mice; and representative immunohistochemical stain for CD31 in sections of hearts from 8-week-old Slit3−/− and WT mice (n = 3 per group). (B) Transcript levels of Slit3, Col1a1, and Col3a1 in the left ventricle and right ventricle in 8-week-old Slit3−/− and WT mice (n = 4–6 per group). (C) Linear regression analysis between the transcript levels of Slit3 and Col1a1 in the left ventricle and right ventricle from 8-week-old WT mice (n = 6 per group). (D) Transcript levels of Slit3 in the left and right ventricle, and their linear regression analysis in 8-week-old WT mice (n = 6 per group). (E) Quantification of perivascular collagen area of coronary arteries and mitral ratio of peak early to late diastolic filling velocity (mitral E/A ratio) in 8-week-old and 1-year-old Slit3−/− and WT mice (n = 4–16 per group). (F) Quantification of cardiac collagen content by assessment of hydroxyproline concentrations in 8-week-old Slit3−/− and WT mice (female, n = 8 per group). (G) Representative passive LV pressure-volume curve and quantification of energy density required to rupture the cardioplegia-relaxed left ventricles by balloon catheter inflation in 8-week-old Slit3−/− and WT mice (n = 4 per group). (H) Representative image of myocardial rupture and overall survival curve of the first week after left anterior descending coronary artery ligation (myocardial infarction, MI) in 8-week-old Slit3−/− and WT mice. The blue arrowhead indicates the position of the myocardial rupture hole, and survival analysis was performed using the Kaplan-Meier method. Log-rank test, P = 0.0039 (n = 24–25 per group). (I) Representative image of femurs or hearts, as well as quan- tification of femur or heart weight/tibia length ratio in 8-week-old Slit3−/− and WT mice (n = 6 per group). Scale bar: 1 mm. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. WT mice using the unpaired 2-tailed Student’s t test (B, E–G, and I). n, number; M, male; F, female.

Techniques: Staining, Immunofluorescence, Immunohistochemical staining, Ligation

$Figure 4. SLIT3 deficiency attenuates LV fibrosis and adverse remodeling. (A) Histological transverse sections of whole heart (first row, Masson’s trichrome stain), coronary arteries (second row, Masson’s trichrome stain), and LV myocytes (third row, hematoxylin and eosin stain) in Slit3−/− and WT mice before and at 3 and 8 weeks after transverse aortic constriction (TAC) (n = 6). Scale bars: 2 mm, 60 or 200 μm, and 60 μm from top to bottom. (B) Quantification of heart weight/tibia length ratio, coronary perivascular fibrosis area, and cardiomyocyte cross-sectional area in Slit3−/− and WT mice before and at 1, 3, and 8 weeks after TAC (n = 4–9 per group). (C) TAC peak pressure gradient determined by echocardiography in Slit3−/− and WT mice after surgery (n = 4–24 per group, initial gradients at day 3, 43 ± 6.8 vs. 43 ± 11 mmHg, P > 0.99). (D and E) Transcript levels of Col1a1, Nppb, and Slit3 in the left ventricle in Slit3−/− and WT mice before and at 1 and 3 weeks after TAC (n = 6–8 per group). (F) LV ejection fraction (EF) determined by echocardiography in Slit3−/− and WT mice before and at 3, 7, and 16 weeks after TAC (n = 5–19 per group). (G) Long-term overall survival curve of Slit3−/− and WT mice from day 1 after TAC. Survival analysis was performed using the Kaplan-Meier method. Log-rank test, P = 0.0245 (n = 25–30 per group). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. WT mice using the unpaired 2-tailed Student’s t test (B–D and F) and 1-way ANOVA with Tamhane T2 multiple comparisons test (E). BSL, 7- to 9-week-old baseline mice before surgery.$

Journal: JCI insight

Article Title: SLIT3 deficiency attenuates pressure overload-induced cardiac fibrosis and remodeling.

doi: 10.1172/jci.insight.136852

Figure Lengend Snippet: Figure 4. SLIT3 deficiency attenuates LV fibrosis and adverse remodeling. (A) Histological transverse sections of whole heart (first row, Masson’s trichrome stain), coronary arteries (second row, Masson’s trichrome stain), and LV myocytes (third row, hematoxylin and eosin stain) in Slit3−/− and WT mice before and at 3 and 8 weeks after transverse aortic constriction (TAC) (n = 6). Scale bars: 2 mm, 60 or 200 μm, and 60 μm from top to bottom. (B) Quantification of heart weight/tibia length ratio, coronary perivascular fibrosis area, and cardiomyocyte cross-sectional area in Slit3−/− and WT mice before and at 1, 3, and 8 weeks after TAC (n = 4–9 per group). (C) TAC peak pressure gradient determined by echocardiography in Slit3−/− and WT mice after surgery (n = 4–24 per group, initial gradients at day 3, 43 ± 6.8 vs. 43 ± 11 mmHg, P > 0.99). (D and E) Transcript levels of Col1a1, Nppb, and Slit3 in the left ventricle in Slit3−/− and WT mice before and at 1 and 3 weeks after TAC (n = 6–8 per group). (F) LV ejection fraction (EF) determined by echocardiography in Slit3−/− and WT mice before and at 3, 7, and 16 weeks after TAC (n = 5–19 per group). (G) Long-term overall survival curve of Slit3−/− and WT mice from day 1 after TAC. Survival analysis was performed using the Kaplan-Meier method. Log-rank test, P = 0.0245 (n = 25–30 per group). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. WT mice using the unpaired 2-tailed Student’s t test (B–D and F) and 1-way ANOVA with Tamhane T2 multiple comparisons test (E). BSL, 7- to 9-week-old baseline mice before surgery.

Techniques: Staining, H&E Stain

$Figure 5. SLIT3 deficiency attenuates RV fibrosis and adverse remodeling. (A) Histological transverse sections of whole heart or part of RV free wall (first row, Masson’s trichrome stain), and RV free wall with coronary arteries (second row, Masson’s trichrome stain), RV free wall myocytes (third row, hema- toxylin and eosin stain) and representative images of RV echocardiogram (fourth row, M-mode parasternal short axis) in Slit3−/− and WT mice before and at 2 and 4 weeks after pulmonary artery banding (PAB) (n = 6). Scale bars: 2 mm, 200 μm, 60 μm, and 2 mm from top to bottom. (B–D) Quantification of RV free wall fibrosis area, myocyte cross-sectional area, as well as end-diastolic diameter area and ratio determined by echocardiogram in Slit3−/− and WT mice before and at 2 and 4 weeks after PAB (n = 4–9 per group). (E) Quantification of PAB peak pressure gradient by echocardiography in Slit3−/− and WT mice at 3, 14, and 28 days after PAB (n = 5–13 per group; initial gradients at day 3, 30 ± 7.9 vs. 29 ± 9.7 mmHg; P > 0.75). (F and G) Transcript levels of Col1a1, Nppb, and Slit3 in the right ventricle in Slit3−/− and WT mice before and at 2 and 4 weeks after PAB (n = 5–6 per group). (H) RV fractional shortening (FS) deter- mined by echocardiography in Slit3−/− and WT mice at 2 weeks after PAB (n = 5–11 per group). (I) Long-term overall survival curve of Slit3−/− and WT mice from day 1 after PAB. Survival analysis was performed using the Kaplan-Meier method. Log-rank test, P = 0.0494 (n = 18–19 per group). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. WT mice using the unpaired 2-tailed Student’s t test (B, E, and F), 1-way ANOVA with Tamhane T2 multiple comparisons test (G), or 2-way ANOVA with Tukey’s multiple comparisons test (C and D). BSL, 7- to 9-week-old baseline mice before surgery.$

Journal: JCI insight

Article Title: SLIT3 deficiency attenuates pressure overload-induced cardiac fibrosis and remodeling.

doi: 10.1172/jci.insight.136852

Figure Lengend Snippet: Figure 5. SLIT3 deficiency attenuates RV fibrosis and adverse remodeling. (A) Histological transverse sections of whole heart or part of RV free wall (first row, Masson’s trichrome stain), and RV free wall with coronary arteries (second row, Masson’s trichrome stain), RV free wall myocytes (third row, hema- toxylin and eosin stain) and representative images of RV echocardiogram (fourth row, M-mode parasternal short axis) in Slit3−/− and WT mice before and at 2 and 4 weeks after pulmonary artery banding (PAB) (n = 6). Scale bars: 2 mm, 200 μm, 60 μm, and 2 mm from top to bottom. (B–D) Quantification of RV free wall fibrosis area, myocyte cross-sectional area, as well as end-diastolic diameter area and ratio determined by echocardiogram in Slit3−/− and WT mice before and at 2 and 4 weeks after PAB (n = 4–9 per group). (E) Quantification of PAB peak pressure gradient by echocardiography in Slit3−/− and WT mice at 3, 14, and 28 days after PAB (n = 5–13 per group; initial gradients at day 3, 30 ± 7.9 vs. 29 ± 9.7 mmHg; P > 0.75). (F and G) Transcript levels of Col1a1, Nppb, and Slit3 in the right ventricle in Slit3−/− and WT mice before and at 2 and 4 weeks after PAB (n = 5–6 per group). (H) RV fractional shortening (FS) deter- mined by echocardiography in Slit3−/− and WT mice at 2 weeks after PAB (n = 5–11 per group). (I) Long-term overall survival curve of Slit3−/− and WT mice from day 1 after PAB. Survival analysis was performed using the Kaplan-Meier method. Log-rank test, P = 0.0494 (n = 18–19 per group). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. WT mice using the unpaired 2-tailed Student’s t test (B, E, and F), 1-way ANOVA with Tamhane T2 multiple comparisons test (G), or 2-way ANOVA with Tukey’s multiple comparisons test (C and D). BSL, 7- to 9-week-old baseline mice before surgery.

Techniques: Staining

Figure 6. SLIT3 deficiency inhibits fibroblast biological activity. (A) MTT cell proliferation assay. Slit3−/− and WT aortic adventitial fibroblasts were seeded in 96-well plates (10% FBS medium) and treated with PBS or PDGF-BB (100 ng/mL). Absorbance was measured at 560 nm at 0 and 48 hours after treatments (n = 6–14 per group). (B) Floating collagen gel contraction assay. Representative images and area quantification of collagen gel. Slit3−/− and WT lung fibroblasts were seeded in PBS or TGF-β1 (5.0 ng/mL) added to collagen gel (1 mg/mL collagen type I, 0.5% FBS). The final to initial area ratio was determined at 24 hours after floating (n = 3 per group). Scale bars: 5 mm. (C) Scratch wound healing assay. Representative images and migration distance quantification of scratch wound healing. Slit3−/− and WT lung fibroblasts were seeded in 6-well plates (1% FBS medium) and treated with PBS or PDGF-BB (100 ng/mL). The migration distance was determined at 24 hours after scratching (n = 4). Scale bars: 20 μm. (D) Excisional wound healing assay. Represen- tative gross and histological images (hematoxylin and eosin staining) and quantification of wound area in 8-week-old Slit3−/− and WT mice at the day of surgery (day 0) and 15 days (day 15) after surgery (n = 19–21 per group). Scale bars: 1 mm and 700 μm from top to bottom. Data are presented as mean ± SD. In vitro experiments were performed at least 3 times independently. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. WT mice or cells using the unpaired 2-tailed Student’s t test (D) or 2-way ANOVA with 2-stage step-up method of Benjamini, Krieger, and Yekutieli multiple comparisons test (A–C).

Journal: JCI insight

Article Title: SLIT3 deficiency attenuates pressure overload-induced cardiac fibrosis and remodeling.

doi: 10.1172/jci.insight.136852

Figure Lengend Snippet: Figure 6. SLIT3 deficiency inhibits fibroblast biological activity. (A) MTT cell proliferation assay. Slit3−/− and WT aortic adventitial fibroblasts were seeded in 96-well plates (10% FBS medium) and treated with PBS or PDGF-BB (100 ng/mL). Absorbance was measured at 560 nm at 0 and 48 hours after treatments (n = 6–14 per group). (B) Floating collagen gel contraction assay. Representative images and area quantification of collagen gel. Slit3−/− and WT lung fibroblasts were seeded in PBS or TGF-β1 (5.0 ng/mL) added to collagen gel (1 mg/mL collagen type I, 0.5% FBS). The final to initial area ratio was determined at 24 hours after floating (n = 3 per group). Scale bars: 5 mm. (C) Scratch wound healing assay. Representative images and migration distance quantification of scratch wound healing. Slit3−/− and WT lung fibroblasts were seeded in 6-well plates (1% FBS medium) and treated with PBS or PDGF-BB (100 ng/mL). The migration distance was determined at 24 hours after scratching (n = 4). Scale bars: 20 μm. (D) Excisional wound healing assay. Represen- tative gross and histological images (hematoxylin and eosin staining) and quantification of wound area in 8-week-old Slit3−/− and WT mice at the day of surgery (day 0) and 15 days (day 15) after surgery (n = 19–21 per group). Scale bars: 1 mm and 700 μm from top to bottom. Data are presented as mean ± SD. In vitro experiments were performed at least 3 times independently. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. WT mice or cells using the unpaired 2-tailed Student’s t test (D) or 2-way ANOVA with 2-stage step-up method of Benjamini, Krieger, and Yekutieli multiple comparisons test (A–C).

Techniques: Activity Assay, MTT Cell Proliferation, Collagen Gel Contraction Assay, Wound Healing Assay, Migration, Staining, In Vitro

Figure 7. SLIT3 regulates YAP1 and fibrillar collagen production. (A and B) Representative images and transcript levels of Acta2, Col1a1, Slit3, and Yap1 in adult primary fibroblasts cultured on stiff surfaces (6-well plastic tissue culture plate, 10% FBS) or in soft collagen gel (1 mg/mL collagen type I, 1% FBS) for 24 hours. From left to right, aortic adventitial fibroblasts (AAFs), cardiac fibroblasts (CFs), and lung fibroblasts (LFs) (n = 3 per group). Scale bars: 20 μm and 50 μm from top to bottom. (C) Transcript levels of Yap1/Taz ratio and Ctgf in the left ventricle in Slit3−/− and WT mice before and at 1 week after TAC (n = 6–8 per group). (D and E) Western blots of YAP1 and phospho-YAP1 in Slit3−/− and WT CFs and LFs cultured on a stiff surface (6-well plastic tissue culture plate) with quantification (n = 2). (F) Transcript levels of Yap1 and Ctgf in Slit3−/− and WT AAFs and CFs in soft collagen gel (1 mg/mL collagen type I, 1% FBS) for 24 hours (n = 3 per group). (G) Linear regression analysis between the transcript levels of Slit3 and Yap1 in WT AAFs cultured on stiff surfaces (6-well plastic tissue culture plate, 10% FBS) or in soft collagen gel (1 mg/mL collagen type I, 1% FBS) for 24 hours (R2 = 0.9114, n = 18). (H) Transcript levels of Yap1, Ctgf, and Col1a1 in Slit3−/− LFs cultured in PBS or recombinant SLIT3 (rSLIT3, aa 34–1116, 1 μg/mL) added to collagen gel (1 mg/mL collagen type I, 0.5% FBS) for 24 hours (n = 10 per group). (I) Transcript levels of Slit3 in WT AAFs and CFs cultured in PBS or TGF-β1 (5.0 ng/mL) added to collagen gel (1 mg/mL collagen type I, 0.5% FBS) for 24 hours (n = 3 per group). (J) Transcript levels of Yap1 and Ctgf in Slit3−/− and WT CFs cultured in PBS or TGF-β1 (5.0 ng/mL) added collagen gel (1 mg/mL collagen type I, 0.5% FBS) for 24 hours (n = 3 per group). Data are presented as mean ± SD. In vitro experiments were independently performed at least 3 times unless indicated otherwise. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. WT mice or cells using the unpaired 2-tailed Student’s t test (B, E, F, H, and I) and 2-way ANOVA with Tukey’s multiple comparisons test (C and J).

Journal: JCI insight

Article Title: SLIT3 deficiency attenuates pressure overload-induced cardiac fibrosis and remodeling.

doi: 10.1172/jci.insight.136852

Figure Lengend Snippet: Figure 7. SLIT3 regulates YAP1 and fibrillar collagen production. (A and B) Representative images and transcript levels of Acta2, Col1a1, Slit3, and Yap1 in adult primary fibroblasts cultured on stiff surfaces (6-well plastic tissue culture plate, 10% FBS) or in soft collagen gel (1 mg/mL collagen type I, 1% FBS) for 24 hours. From left to right, aortic adventitial fibroblasts (AAFs), cardiac fibroblasts (CFs), and lung fibroblasts (LFs) (n = 3 per group). Scale bars: 20 μm and 50 μm from top to bottom. (C) Transcript levels of Yap1/Taz ratio and Ctgf in the left ventricle in Slit3−/− and WT mice before and at 1 week after TAC (n = 6–8 per group). (D and E) Western blots of YAP1 and phospho-YAP1 in Slit3−/− and WT CFs and LFs cultured on a stiff surface (6-well plastic tissue culture plate) with quantification (n = 2). (F) Transcript levels of Yap1 and Ctgf in Slit3−/− and WT AAFs and CFs in soft collagen gel (1 mg/mL collagen type I, 1% FBS) for 24 hours (n = 3 per group). (G) Linear regression analysis between the transcript levels of Slit3 and Yap1 in WT AAFs cultured on stiff surfaces (6-well plastic tissue culture plate, 10% FBS) or in soft collagen gel (1 mg/mL collagen type I, 1% FBS) for 24 hours (R2 = 0.9114, n = 18). (H) Transcript levels of Yap1, Ctgf, and Col1a1 in Slit3−/− LFs cultured in PBS or recombinant SLIT3 (rSLIT3, aa 34–1116, 1 μg/mL) added to collagen gel (1 mg/mL collagen type I, 0.5% FBS) for 24 hours (n = 10 per group). (I) Transcript levels of Slit3 in WT AAFs and CFs cultured in PBS or TGF-β1 (5.0 ng/mL) added to collagen gel (1 mg/mL collagen type I, 0.5% FBS) for 24 hours (n = 3 per group). (J) Transcript levels of Yap1 and Ctgf in Slit3−/− and WT CFs cultured in PBS or TGF-β1 (5.0 ng/mL) added collagen gel (1 mg/mL collagen type I, 0.5% FBS) for 24 hours (n = 3 per group). Data are presented as mean ± SD. In vitro experiments were independently performed at least 3 times unless indicated otherwise. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. WT mice or cells using the unpaired 2-tailed Student’s t test (B, E, F, H, and I) and 2-way ANOVA with Tukey’s multiple comparisons test (C and J).

Techniques: Cell Culture, Western Blot, Recombinant, In Vitro

( A ) Pie charts displaying the genome-wide distribution (left) and promoter distal distribution (right) of DNTTIP1 in WT mESCs. DNTTIP1 peaks within 1 kb of the transcription start site (TSS) were assigned to TSS. ( B ) Venn Diagram depicting the overlap between DNTTIP1, H3K4me1 and H3K27ac peaks in WT mESCs. ( C ) Venn diagram showing the number of downregulated and upregulated genes in Dnttip1 KO (KO1 and KO2) versus WT mESCs that are bound by DNTTIP1 in WT mESCs. ( D ) Heatmaps displaying the genome-wide distribution of all DNTTIP1 binding sites in WT, Dnttip1 KO1 and Elmsan1 KO1 mESCs sorted by enrichment in descending order in WT mESCs and compared to HDAC1 occupancy in WT mESCs. The color scale depicts the normalized ChIP-seq signal intensity (log 2 CPM per 20 bp bin). ( E ) Venn diagram showing the co-occupancy between DNTTIP1 and HDAC1 peaks in WT mESCs. ( A–E ) DNTTIP1, H3K4me1 and H3K27ac ChIP-seq peaks were determined based on the average of two replicates each from WT mESCs and where applicable based on the average of two replicates each from Dnttip1 KO1 and Elmsan1 KO1 mESCs, respectively. HDAC1 ChIP-seq data were obtained from GSE55437. Downregulated and upregulated genes were determined based on two biological replicates each from WT mESCs and two Dnttip1 KO (KO1 and KO2) clones. ( F, G ) ChIP-seq profiles of the ( F ) Slit3 and ( G ) Spry4 loci for DNTTIP1 in WT, Dnttip1 KO1 and Elmsan1 KO1 mESCs and for HDAC1 in WT mESCs. Promoter and putative enhancer regions used for manual ChIP experiments in are highlighted by orange boxes. The track files depict the average of two ChIP-seq replicates each from WT mESCs and the average of two replicates each from the Dnttip1 KO1 and Elmsan1 KO1 clone, respectively. ( H ) WB for the specified histone acetylation marks from total cell lysates of WT, Dnttip1 KO1 and Elmsan1 KO1 mESCs. H3 and H4 are loading controls.

Journal: eLife

Article Title: The histone deacetylase complex MiDAC regulates a neurodevelopmental gene expression program to control neurite outgrowth

doi: 10.7554/eLife.57519

Figure Lengend Snippet: ( A ) Pie charts displaying the genome-wide distribution (left) and promoter distal distribution (right) of DNTTIP1 in WT mESCs. DNTTIP1 peaks within 1 kb of the transcription start site (TSS) were assigned to TSS. ( B ) Venn Diagram depicting the overlap between DNTTIP1, H3K4me1 and H3K27ac peaks in WT mESCs. ( C ) Venn diagram showing the number of downregulated and upregulated genes in Dnttip1 KO (KO1 and KO2) versus WT mESCs that are bound by DNTTIP1 in WT mESCs. ( D ) Heatmaps displaying the genome-wide distribution of all DNTTIP1 binding sites in WT, Dnttip1 KO1 and Elmsan1 KO1 mESCs sorted by enrichment in descending order in WT mESCs and compared to HDAC1 occupancy in WT mESCs. The color scale depicts the normalized ChIP-seq signal intensity (log 2 CPM per 20 bp bin). ( E ) Venn diagram showing the co-occupancy between DNTTIP1 and HDAC1 peaks in WT mESCs. ( A–E ) DNTTIP1, H3K4me1 and H3K27ac ChIP-seq peaks were determined based on the average of two replicates each from WT mESCs and where applicable based on the average of two replicates each from Dnttip1 KO1 and Elmsan1 KO1 mESCs, respectively. HDAC1 ChIP-seq data were obtained from GSE55437. Downregulated and upregulated genes were determined based on two biological replicates each from WT mESCs and two Dnttip1 KO (KO1 and KO2) clones. ( F, G ) ChIP-seq profiles of the ( F ) Slit3 and ( G ) Spry4 loci for DNTTIP1 in WT, Dnttip1 KO1 and Elmsan1 KO1 mESCs and for HDAC1 in WT mESCs. Promoter and putative enhancer regions used for manual ChIP experiments in are highlighted by orange boxes. The track files depict the average of two ChIP-seq replicates each from WT mESCs and the average of two replicates each from the Dnttip1 KO1 and Elmsan1 KO1 clone, respectively. ( H ) WB for the specified histone acetylation marks from total cell lysates of WT, Dnttip1 KO1 and Elmsan1 KO1 mESCs. H3 and H4 are loading controls.

Article Snippet: However, instead of co-culturing GNPs with Dnttip1 KO NE, GNPs were supplemented daily with CM of Dnttip1 KO NE (50%), 50% fresh differentiation medium 3 and recombinant SLIT3 (R&D Systems, 9296-SL-050), NTN1 (R&D Systems, 6419-N1-025) or a combination of SLIT3/NTN1 for 6 days from the day of GNP seeding.

Techniques: Genome Wide, Binding Assay, ChIP-sequencing, Clone Assay

( A–J ) qPCR from manual ChIP experiments against ( A, F ) DNTTIP1, ( B, G ) ELMSAN1, ( C, H ) HDAC1, ( D, I ) H3K27ac and ( E, J ) H4K20ac from WT, Dnttip1 KO1 and Elmsan1 KO1 NE targeting select promoter, putative enhancer and intragenic control regions of ( A–E ) Slit3 or ( F-J ) Spry4 loci as highlighted in . IgG was used as a control antibody. Unpaired t-test was performed throughout where **, p≤0.01; and ns, p>0.05 is not significant.

Journal: eLife

Article Title: The histone deacetylase complex MiDAC regulates a neurodevelopmental gene expression program to control neurite outgrowth

doi: 10.7554/eLife.57519

Figure Lengend Snippet: ( A–J ) qPCR from manual ChIP experiments against ( A, F ) DNTTIP1, ( B, G ) ELMSAN1, ( C, H ) HDAC1, ( D, I ) H3K27ac and ( E, J ) H4K20ac from WT, Dnttip1 KO1 and Elmsan1 KO1 NE targeting select promoter, putative enhancer and intragenic control regions of ( A–E ) Slit3 or ( F-J ) Spry4 loci as highlighted in . IgG was used as a control antibody. Unpaired t-test was performed throughout where **, p≤0.01; and ns, p>0.05 is not significant.

Techniques: Control

( A ) WB for signaling components of the SLIT3/ROBO3 and NTN1/DCC/UNC5B signaling axes from CM and total cell lysates of WT and Dnttip1 KO1 NE after 12 days of differentiation. To enrich SLIT3 and NTN1 from CM, IPs were performed with SLIT3 and NTN1 antibodies from CM of WT and Dnttip1 KO1 NE. Actin is the loading control for the total cell lysates. ( B ) Assay to rescue the neurite outgrowth defects in Dnttip1 KO1 NE. CM of Dnttip1 KO1 NE was supplemented with the recombinant signaling ligands SLIT3 and/or NTN1 from day 7–12 without or with preblocking of Dnttip1 KO1 NE with IgG or signaling receptor antibodies against ROBO3 and/or UNC5B. MAP2 IF staining was performed after 12 days of differentiation. Nuclei were stained with DAPI. To facilitate analysis the neuronal cell body (blue) and its neurites were manually traced with ImageJ software and for each sample one traced neuron is displayed in the inlet. The white scale bar represents 50 µm. ( C, D ) Quantification of ( C ) neurite length and ( D ) the total number of neurites per neuron from the MAP2 IF staining in ( B ) using ImageJ. ( C ) Neurite length was divided into two categories of short neurites <50 µm (green box plots) and longer neurites ≥50 µm (white box plots). ( C, D ) The neurites of 200 neurons were assessed per sample. One-way ANOVA was performed throughout where ***, p≤0.001; and ns, p>0.05 is not significant.

Journal: eLife

Article Title: The histone deacetylase complex MiDAC regulates a neurodevelopmental gene expression program to control neurite outgrowth

doi: 10.7554/eLife.57519

Figure Lengend Snippet: ( A ) WB for signaling components of the SLIT3/ROBO3 and NTN1/DCC/UNC5B signaling axes from CM and total cell lysates of WT and Dnttip1 KO1 NE after 12 days of differentiation. To enrich SLIT3 and NTN1 from CM, IPs were performed with SLIT3 and NTN1 antibodies from CM of WT and Dnttip1 KO1 NE. Actin is the loading control for the total cell lysates. ( B ) Assay to rescue the neurite outgrowth defects in Dnttip1 KO1 NE. CM of Dnttip1 KO1 NE was supplemented with the recombinant signaling ligands SLIT3 and/or NTN1 from day 7–12 without or with preblocking of Dnttip1 KO1 NE with IgG or signaling receptor antibodies against ROBO3 and/or UNC5B. MAP2 IF staining was performed after 12 days of differentiation. Nuclei were stained with DAPI. To facilitate analysis the neuronal cell body (blue) and its neurites were manually traced with ImageJ software and for each sample one traced neuron is displayed in the inlet. The white scale bar represents 50 µm. ( C, D ) Quantification of ( C ) neurite length and ( D ) the total number of neurites per neuron from the MAP2 IF staining in ( B ) using ImageJ. ( C ) Neurite length was divided into two categories of short neurites <50 µm (green box plots) and longer neurites ≥50 µm (white box plots). ( C, D ) The neurites of 200 neurons were assessed per sample. One-way ANOVA was performed throughout where ***, p≤0.001; and ns, p>0.05 is not significant.

Techniques: Control, Recombinant, Staining, Software

( A ) Chamber assay to rescue the network formation defects of GNP-derived neurons that were supplemented with CM of Dnttip1 KO1 NE. CM of Dnttip1 KO1 NE was supplemented with the recombinant signaling ligands SLIT3 and/or NTN1 from day 7–12 without or with pre-blocking of GNP-derived neurons with control IgG or signaling receptor antibodies against ROBO3 and/or UNC5B. TUBB3 IF staining was performed after 12 days of differentiation. Nuclei were stained with DAPI. The white scale bar represents 100 µm. ( B ) Quantification of neuronal network formation from the TUBB3 IF staining in ( A ) using ImageJ. The percentage of formed neuronal networks within the total population of TUBB3-positive neurons is displayed. A neuronal network was scored when a closed local circuit was detected around an individual neuron. Neuronal network formation was assessed for 100 neurons per sample in triplicate. Unpaired t-test was performed throughout where ***, p≤0.01.

Journal: eLife

Article Title: The histone deacetylase complex MiDAC regulates a neurodevelopmental gene expression program to control neurite outgrowth

doi: 10.7554/eLife.57519

Figure Lengend Snippet: ( A ) Chamber assay to rescue the network formation defects of GNP-derived neurons that were supplemented with CM of Dnttip1 KO1 NE. CM of Dnttip1 KO1 NE was supplemented with the recombinant signaling ligands SLIT3 and/or NTN1 from day 7–12 without or with pre-blocking of GNP-derived neurons with control IgG or signaling receptor antibodies against ROBO3 and/or UNC5B. TUBB3 IF staining was performed after 12 days of differentiation. Nuclei were stained with DAPI. The white scale bar represents 100 µm. ( B ) Quantification of neuronal network formation from the TUBB3 IF staining in ( A ) using ImageJ. The percentage of formed neuronal networks within the total population of TUBB3-positive neurons is displayed. A neuronal network was scored when a closed local circuit was detected around an individual neuron. Neuronal network formation was assessed for 100 neurons per sample in triplicate. Unpaired t-test was performed throughout where ***, p≤0.01.

Techniques: Boyden Chamber Assay, Derivative Assay, Recombinant, Blocking Assay, Control, Staining

MiDAC directly binds to and deacetylates H4K20ac on regulatory elements of pro-neural genes such as those of the axon guidance ligands SLIT3 and NTN1 resulting in the activation of these genes. Conversely, MiDAC inhibits the gene expression of negative regulators of neurogenesis such as SPRY4 and ID1 by binding and removing H3K27ac from their promoters and enhancers. SLIT3 and NTN1, the downstream targets of MiDAC, bind to their cognate receptors ROBO3 and UNC5B respectively thereby activating the signaling cascade responsible for promoting neurite outgrowth.

Journal: eLife

Article Title: The histone deacetylase complex MiDAC regulates a neurodevelopmental gene expression program to control neurite outgrowth

doi: 10.7554/eLife.57519

Figure Lengend Snippet: MiDAC directly binds to and deacetylates H4K20ac on regulatory elements of pro-neural genes such as those of the axon guidance ligands SLIT3 and NTN1 resulting in the activation of these genes. Conversely, MiDAC inhibits the gene expression of negative regulators of neurogenesis such as SPRY4 and ID1 by binding and removing H3K27ac from their promoters and enhancers. SLIT3 and NTN1, the downstream targets of MiDAC, bind to their cognate receptors ROBO3 and UNC5B respectively thereby activating the signaling cascade responsible for promoting neurite outgrowth.

Techniques: Activation Assay, Gene Expression, Binding Assay

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