unc5b Search Results


gene exp unc5b hs00402127 m1  (Thermo Fisher)
85
Thermo Fisher gene exp unc5b hs00402127 m1
Gene Exp Unc5b Hs00402127 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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goat anti unc5b antibody  (R&D Systems)
99
R&D Systems goat anti unc5b antibody
Netrin-1 mobilizes Ca 2+ and drives MMP3 activity in vascular smooth muscle cells via its receptor neogenin-1. a Calcium integration curves in VSMCs stimulation as indicated. Data are expressed as 340/380 nm excitation ratio. n = 4. * P < 0.05. b MMP3 activity in murine VSMCs stimulated with netrin-1 in the presence or absence of calcium chelator BAPTA (10 µM). n = 6–7 per group. ** P < 0.01. c NFATc3 (green) and actin (F-actin, red) immunofluorescence staining in mouse VSMCs (top panel) or human VSMCs (lower panel) stimulated with netrin-1 (2.5 µg/ml) with or without BAPTA. White arrowheads indicate nuclear location, n = 4. Scale bars 20 µm. d MMP3 activity in mouse VSMCs stimulated with netrin-1 in the presence or absence of NFATc3 inhibitor, VIVIT (1 µM). n = 6–7 per group. *** P < 0.001. e Immunofluorescence staining of neogenin-1 (NEO-1, red) and alpha-smooth muscle actin (SMA, green) in aortic sections of ApoE −/− mice. Colocalization is shown in merge. Nuclei stained with DAPI (blue). Scale bar 50 µm. f MMP3 activity in VSMCs stimulated with recombinant netrin-1 in the presence of control IgG, anti-NEO1, or <t>anti-UNC5B</t> neutralizing antibody (10 µg/ml). n = 6 per group. * P < 0.05, NS not significant. g MMP3 activity in VSMCs transfected with siRNA control, Neo1 or Unc5b and stimulated with recombinant netrin-1. n = 4–9 per group. * P < 0.05, ** P < 0.01, NS = not significant. h Calcium integration curves in VSMCs stimulated as indicated. Data are expressed as 340/380 nm excitation ratio. * P < 0.05. i NFATc3 and F-actin immunofluorescence staining of VSMCs stimulated with netrin-1, in the presence of control IgG or anti-NEO1 blocking antibody. White arrowheads indicate nuclear translocations. Scale bar 20 µm. j MMP3 enzymatic activity in VSMCs co-cultured with WTMø in the presence of control IgG or anti-NEO1 antibody. n = 4–8 per group. * P < 0.05. Two-way ANOVA was used to assess statistical significance in a and h . Unpaired, two-tailed t -test was used for statistical analysis in b , d , f , g , and j . Error bars represent s.e.m
Goat Anti Unc5b Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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unc5b  (Cell Signaling Technology Inc)
94
Cell Signaling Technology Inc unc5b
A CRISPR screen identifies <t>UNC5B</t> as a YAP effector. A, Z -scores comparing YAP-expressing (day 25) to Empty (day 25) cells for each sgRNA in the CRISPR screen. Select genes are indicated. n = 4. B, TEAD1 knockout rescues YAP-induced cytostasis in Y79 cells (left). Western blot showing TEAD1 knockout efficiency and expression of ectopic YAP (right). *, P < 0.05 compared with sgControl cells, n = 3. C, UNC5B knockout rescues YAP-induced cytostasis in Y79 cells (left). Western blot showing UNC5B knockout efficiency and expression of ectopic YAP (right). ***, P < 0.001 compared with sgControl cells, n = 4. D, RT-qPCR for YAP target genes ( AJUBA, AMOTL2, CYR61 and AHNAK ) or a control gene ( RER1 ) in control or UNC5B knockout Y79 cells ± ectopic YAP expression. n = 3.
Unc5b, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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gene exp unc5b hs00900710 m1  (Thermo Fisher)
94
Thermo Fisher gene exp unc5b hs00900710 m1
A CRISPR screen identifies <t>UNC5B</t> as a YAP effector. A, Z -scores comparing YAP-expressing (day 25) to Empty (day 25) cells for each sgRNA in the CRISPR screen. Select genes are indicated. n = 4. B, TEAD1 knockout rescues YAP-induced cytostasis in Y79 cells (left). Western blot showing TEAD1 knockout efficiency and expression of ectopic YAP (right). *, P < 0.05 compared with sgControl cells, n = 3. C, UNC5B knockout rescues YAP-induced cytostasis in Y79 cells (left). Western blot showing UNC5B knockout efficiency and expression of ectopic YAP (right). ***, P < 0.001 compared with sgControl cells, n = 4. D, RT-qPCR for YAP target genes ( AJUBA, AMOTL2, CYR61 and AHNAK ) or a control gene ( RER1 ) in control or UNC5B knockout Y79 cells ± ectopic YAP expression. n = 3.
Gene Exp Unc5b Hs00900710 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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gene exp unc5b mm00504054 m1  (Thermo Fisher)
97
Thermo Fisher gene exp unc5b mm00504054 m1
A CRISPR screen identifies <t>UNC5B</t> as a YAP effector. A, Z -scores comparing YAP-expressing (day 25) to Empty (day 25) cells for each sgRNA in the CRISPR screen. Select genes are indicated. n = 4. B, TEAD1 knockout rescues YAP-induced cytostasis in Y79 cells (left). Western blot showing TEAD1 knockout efficiency and expression of ectopic YAP (right). *, P < 0.05 compared with sgControl cells, n = 3. C, UNC5B knockout rescues YAP-induced cytostasis in Y79 cells (left). Western blot showing UNC5B knockout efficiency and expression of ectopic YAP (right). ***, P < 0.001 compared with sgControl cells, n = 4. D, RT-qPCR for YAP target genes ( AJUBA, AMOTL2, CYR61 and AHNAK ) or a control gene ( RER1 ) in control or UNC5B knockout Y79 cells ± ectopic YAP expression. n = 3.
Gene Exp Unc5b Mm00504054 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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gene exp unc5b as1 hs04274416 g1  (Thermo Fisher)
91
Thermo Fisher gene exp unc5b as1 hs04274416 g1
Primer sequences used for real-time PCR.
Gene Exp Unc5b As1 Hs04274416 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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unc5b  (R&D Systems)
92
R&D Systems unc5b
( A, B ) ChIP-seq profiles of the ( A ) Robo3 and ( B ) <t>Unc5b</t> loci for DNTTIP1 in WT, Dnttip1 KO1 and Elmsan1 KO1 mESCs and for HDAC1 in WT mESCs. Promoter regions used for manual ChIP experiments in ( C ) and ( E ) are highlighted by orange boxes. DNTTIP1 ChIP-seq peaks were determined based on the average of two replicates each from WT mESCs and based on the average of two replicates each from the Dnttip1 KO1 and Elmsan1 KO1 clone, respectively. HDAC1 ChIP-seq data were obtained from GSE55437. ( C ) qPCR from manual ChIP experiments against DNTTIP1, ELMSAN1 and HDAC1 from WT, Dnttip1 KO1 and Elmsan1 KO1 NE targeting the promoter of the Robo3 locus as highlighted in ( A ). IgG was used as a control antibody. ( D ) qRT-PCR for Robo3 mRNA in WT, Dnttip1 KO1 and Elmsan1 KO1 NE after 12 days of differentiation. Expression was normalized to Gapdh. ( E ) qPCR from manual ChIP experiments against DNTTIP1, ELMSAN1 and HDAC1 from WT, Dnttip1 KO1 and Elmsan1 KO1 NE targeting the promoter of the Unc5b locus as highlighted in ( B ). IgG was used as a control antibody. ( F ) qRT-PCR for Unc5b mRNA in WT, Dnttip1 KO1 and Elmsan1 KO1 NE after 12 days of differentiation. Expression was normalized to Gapdh .
Unc5b, supplied by R&D Systems, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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unc5b  (Bioss)
90
Bioss unc5b
( A, B ) ChIP-seq profiles of the ( A ) Robo3 and ( B ) <t>Unc5b</t> loci for DNTTIP1 in WT, Dnttip1 KO1 and Elmsan1 KO1 mESCs and for HDAC1 in WT mESCs. Promoter regions used for manual ChIP experiments in ( C ) and ( E ) are highlighted by orange boxes. DNTTIP1 ChIP-seq peaks were determined based on the average of two replicates each from WT mESCs and based on the average of two replicates each from the Dnttip1 KO1 and Elmsan1 KO1 clone, respectively. HDAC1 ChIP-seq data were obtained from GSE55437. ( C ) qPCR from manual ChIP experiments against DNTTIP1, ELMSAN1 and HDAC1 from WT, Dnttip1 KO1 and Elmsan1 KO1 NE targeting the promoter of the Robo3 locus as highlighted in ( A ). IgG was used as a control antibody. ( D ) qRT-PCR for Robo3 mRNA in WT, Dnttip1 KO1 and Elmsan1 KO1 NE after 12 days of differentiation. Expression was normalized to Gapdh. ( E ) qPCR from manual ChIP experiments against DNTTIP1, ELMSAN1 and HDAC1 from WT, Dnttip1 KO1 and Elmsan1 KO1 NE targeting the promoter of the Unc5b locus as highlighted in ( B ). IgG was used as a control antibody. ( F ) qRT-PCR for Unc5b mRNA in WT, Dnttip1 KO1 and Elmsan1 KO1 NE after 12 days of differentiation. Expression was normalized to Gapdh .
Unc5b, supplied by Bioss, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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gene exp unc5b rn00573551 m1  (Thermo Fisher)
90
Thermo Fisher gene exp unc5b rn00573551 m1
(A and B) Immunofluorescence staining for Netrin-4 (A) and <t>Unc5B</t> (B) in Vc of the ipsilateral side and contralateral side. Bar:100μm. (C and D) Netrin-4 (green) and NeuN (red) double immunofluorescence staining in Vc of the ipsilateral side. Bar: (C) 100μm (D) 10μm. (E and F) Unc5B (green) and NeuN (red) double immunofluorescence staining in Vc of the ipsilateral side. Bar: (E) 100μm (F) 10μm. (G and H) Netrin-4 (G) and Unc5B (F) mRNA expression in the ipsilateral (ipsi) and contralateral (contra) side at 14days after CCI-Sham injury (n = 3–5). The right graphs show the other day time results. The data are presented as the mean ± SEM. *, P<0.05; Mann-Whitney test.
Gene Exp Unc5b Rn00573551 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 90 stars, based on 1 article reviews
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human unc5b cdna  (Sino Biological)
91
Sino Biological human unc5b cdna
<t>UNC5B</t> mediates FLRT2-promoted THP-1 cell maturation into macrophages. FLRT2 was overexpressed by transfecting the hFLRT2 vector, and UNC5B was silenced by transfecting UNC5B-specific shRNA into THP-1 cells. (A) Representative phase-contrast light microscopy images showing morphological alterations of THP-1 cells transfected as indicated (n = 3). Scale bar, 50 μm. (B) qPCR analysis of Itgam , Csf1r , Cd36 , Msr1 , and Olr1 mRNA in THP-1 cells transfected as indicated (n = 3). (C) Immunoblot analysis of CD36, SR-A, UNC5B, and GFP-FLRT2 proteins in THP-1 cells transfected as indicated (n = 3). (D) HUVECs expressing RFP were co-cultured with THP-1 cells transfected as indicated. After 6 h co-culture, pictures were taken using a fluorescence microscope (n = 3). Scale bar, 5 mm. (E) Transwell cell migration assay was performed in THP-1 cells transfected as indicated, and the numbers of the migrated cells were counted (n = 3). Scale bar, 100 μm. (F) Phagocytosis assays were performed by culturing the cells transfected as indicated in Texas red-conjugated zymosan particles for 2 h at 37°C (n = 3). Cells were observed for internalization of the particles by fluorescence microscopy. Scale bar, 50 μm. Data are means ± SD. P values were determined using unpaired, two-tailed Student’s t -tests. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.
Human Unc5b Cdna, supplied by Sino Biological, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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peptides  (Addgene inc)
90
Addgene inc peptides
<t>UNC5B</t> mediates FLRT2-promoted THP-1 cell maturation into macrophages. FLRT2 was overexpressed by transfecting the hFLRT2 vector, and UNC5B was silenced by transfecting UNC5B-specific shRNA into THP-1 cells. (A) Representative phase-contrast light microscopy images showing morphological alterations of THP-1 cells transfected as indicated (n = 3). Scale bar, 50 μm. (B) qPCR analysis of Itgam , Csf1r , Cd36 , Msr1 , and Olr1 mRNA in THP-1 cells transfected as indicated (n = 3). (C) Immunoblot analysis of CD36, SR-A, UNC5B, and GFP-FLRT2 proteins in THP-1 cells transfected as indicated (n = 3). (D) HUVECs expressing RFP were co-cultured with THP-1 cells transfected as indicated. After 6 h co-culture, pictures were taken using a fluorescence microscope (n = 3). Scale bar, 5 mm. (E) Transwell cell migration assay was performed in THP-1 cells transfected as indicated, and the numbers of the migrated cells were counted (n = 3). Scale bar, 100 μm. (F) Phagocytosis assays were performed by culturing the cells transfected as indicated in Texas red-conjugated zymosan particles for 2 h at 37°C (n = 3). Cells were observed for internalization of the particles by fluorescence microscopy. Scale bar, 50 μm. Data are means ± SD. P values were determined using unpaired, two-tailed Student’s t -tests. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.
Peptides, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


Netrin-1 mobilizes Ca 2+ and drives MMP3 activity in vascular smooth muscle cells via its receptor neogenin-1. a Calcium integration curves in VSMCs stimulation as indicated. Data are expressed as 340/380 nm excitation ratio. n = 4. * P < 0.05. b MMP3 activity in murine VSMCs stimulated with netrin-1 in the presence or absence of calcium chelator BAPTA (10 µM). n = 6–7 per group. ** P < 0.01. c NFATc3 (green) and actin (F-actin, red) immunofluorescence staining in mouse VSMCs (top panel) or human VSMCs (lower panel) stimulated with netrin-1 (2.5 µg/ml) with or without BAPTA. White arrowheads indicate nuclear location, n = 4. Scale bars 20 µm. d MMP3 activity in mouse VSMCs stimulated with netrin-1 in the presence or absence of NFATc3 inhibitor, VIVIT (1 µM). n = 6–7 per group. *** P < 0.001. e Immunofluorescence staining of neogenin-1 (NEO-1, red) and alpha-smooth muscle actin (SMA, green) in aortic sections of ApoE −/− mice. Colocalization is shown in merge. Nuclei stained with DAPI (blue). Scale bar 50 µm. f MMP3 activity in VSMCs stimulated with recombinant netrin-1 in the presence of control IgG, anti-NEO1, or anti-UNC5B neutralizing antibody (10 µg/ml). n = 6 per group. * P < 0.05, NS not significant. g MMP3 activity in VSMCs transfected with siRNA control, Neo1 or Unc5b and stimulated with recombinant netrin-1. n = 4–9 per group. * P < 0.05, ** P < 0.01, NS = not significant. h Calcium integration curves in VSMCs stimulated as indicated. Data are expressed as 340/380 nm excitation ratio. * P < 0.05. i NFATc3 and F-actin immunofluorescence staining of VSMCs stimulated with netrin-1, in the presence of control IgG or anti-NEO1 blocking antibody. White arrowheads indicate nuclear translocations. Scale bar 20 µm. j MMP3 enzymatic activity in VSMCs co-cultured with WTMø in the presence of control IgG or anti-NEO1 antibody. n = 4–8 per group. * P < 0.05. Two-way ANOVA was used to assess statistical significance in a and h . Unpaired, two-tailed t -test was used for statistical analysis in b , d , f , g , and j . Error bars represent s.e.m

Journal: Nature Communications

Article Title: Macrophage-derived netrin-1 promotes abdominal aortic aneurysm formation by activating MMP3 in vascular smooth muscle cells

doi: 10.1038/s41467-018-07495-1

Figure Lengend Snippet: Netrin-1 mobilizes Ca 2+ and drives MMP3 activity in vascular smooth muscle cells via its receptor neogenin-1. a Calcium integration curves in VSMCs stimulation as indicated. Data are expressed as 340/380 nm excitation ratio. n = 4. * P < 0.05. b MMP3 activity in murine VSMCs stimulated with netrin-1 in the presence or absence of calcium chelator BAPTA (10 µM). n = 6–7 per group. ** P < 0.01. c NFATc3 (green) and actin (F-actin, red) immunofluorescence staining in mouse VSMCs (top panel) or human VSMCs (lower panel) stimulated with netrin-1 (2.5 µg/ml) with or without BAPTA. White arrowheads indicate nuclear location, n = 4. Scale bars 20 µm. d MMP3 activity in mouse VSMCs stimulated with netrin-1 in the presence or absence of NFATc3 inhibitor, VIVIT (1 µM). n = 6–7 per group. *** P < 0.001. e Immunofluorescence staining of neogenin-1 (NEO-1, red) and alpha-smooth muscle actin (SMA, green) in aortic sections of ApoE −/− mice. Colocalization is shown in merge. Nuclei stained with DAPI (blue). Scale bar 50 µm. f MMP3 activity in VSMCs stimulated with recombinant netrin-1 in the presence of control IgG, anti-NEO1, or anti-UNC5B neutralizing antibody (10 µg/ml). n = 6 per group. * P < 0.05, NS not significant. g MMP3 activity in VSMCs transfected with siRNA control, Neo1 or Unc5b and stimulated with recombinant netrin-1. n = 4–9 per group. * P < 0.05, ** P < 0.01, NS = not significant. h Calcium integration curves in VSMCs stimulated as indicated. Data are expressed as 340/380 nm excitation ratio. * P < 0.05. i NFATc3 and F-actin immunofluorescence staining of VSMCs stimulated with netrin-1, in the presence of control IgG or anti-NEO1 blocking antibody. White arrowheads indicate nuclear translocations. Scale bar 20 µm. j MMP3 enzymatic activity in VSMCs co-cultured with WTMø in the presence of control IgG or anti-NEO1 antibody. n = 4–8 per group. * P < 0.05. Two-way ANOVA was used to assess statistical significance in a and h . Unpaired, two-tailed t -test was used for statistical analysis in b , d , f , g , and j . Error bars represent s.e.m

Article Snippet: In some assays, neutralizing antibodies, goat anti-neogenin-1 antibody (AF1079-SP, R&D Systems), goat anti-Unc5b antibody (AF1006-SP, R&D Systems) or goat IgG control (AB-108-C, R&D Systems) were added 30 min prior to stimulations.

Techniques: Activity Assay, Immunofluorescence, Staining, Recombinant, Control, Transfection, Blocking Assay, Cell Culture, Two Tailed Test

A CRISPR screen identifies UNC5B as a YAP effector. A, Z -scores comparing YAP-expressing (day 25) to Empty (day 25) cells for each sgRNA in the CRISPR screen. Select genes are indicated. n = 4. B, TEAD1 knockout rescues YAP-induced cytostasis in Y79 cells (left). Western blot showing TEAD1 knockout efficiency and expression of ectopic YAP (right). *, P < 0.05 compared with sgControl cells, n = 3. C, UNC5B knockout rescues YAP-induced cytostasis in Y79 cells (left). Western blot showing UNC5B knockout efficiency and expression of ectopic YAP (right). ***, P < 0.001 compared with sgControl cells, n = 4. D, RT-qPCR for YAP target genes ( AJUBA, AMOTL2, CYR61 and AHNAK ) or a control gene ( RER1 ) in control or UNC5B knockout Y79 cells ± ectopic YAP expression. n = 3.

Journal: Cancer Research Communications

Article Title: Netrin-1 and UNC5B Cooperate with Integrins to Mediate YAP-Driven Cytostasis

doi: 10.1158/2767-9764.CRC-24-0101

Figure Lengend Snippet: A CRISPR screen identifies UNC5B as a YAP effector. A, Z -scores comparing YAP-expressing (day 25) to Empty (day 25) cells for each sgRNA in the CRISPR screen. Select genes are indicated. n = 4. B, TEAD1 knockout rescues YAP-induced cytostasis in Y79 cells (left). Western blot showing TEAD1 knockout efficiency and expression of ectopic YAP (right). *, P < 0.05 compared with sgControl cells, n = 3. C, UNC5B knockout rescues YAP-induced cytostasis in Y79 cells (left). Western blot showing UNC5B knockout efficiency and expression of ectopic YAP (right). ***, P < 0.001 compared with sgControl cells, n = 4. D, RT-qPCR for YAP target genes ( AJUBA, AMOTL2, CYR61 and AHNAK ) or a control gene ( RER1 ) in control or UNC5B knockout Y79 cells ± ectopic YAP expression. n = 3.

Article Snippet: The following antibodies were used: YAP/TAZ (Santa Cruz Biotech, sc-101199; RRID: AB_1131430), GFP (Santa Cruz Biotech, sc-9996; RRID: AB_627695), TUBULIN (Santa Cruz Biotech, sc-32293; RRID: AB_628412), TEAD1 (BD Biosciences, 610923; RRID: AB_398238), and UNC5B (Cell Signaling Technology, 13851; RRID:AB_2798330).

Techniques: CRISPR, Expressing, Knock-Out, Western Blot, Quantitative RT-PCR, Control

YAP induces Netrin and UNC5 family members in YAP off cancers. A–F, UNC5B Western blots from YAP off cells ectopically expressing YAP or YAP mutants ( A , C , E , F ) or a TEAD4 DNA-binding-domain (DBD)-VP64 fusion protein or controls ( B , D ). Cell lines and tumor types are indicated in each. n = 3. G, UNC5B Western blot from YAP off and YAP on cell lines. n = 2. H, Heatmap showing the effect of ectopic YAP on the expression of UNC5A-D or Netrins (NTN1, 2 and 4) in YAP off cells. * FDR < 0.05; N = not detected. RNA expression levels mined from RNA-Seq data in .

Journal: Cancer Research Communications

Article Title: Netrin-1 and UNC5B Cooperate with Integrins to Mediate YAP-Driven Cytostasis

doi: 10.1158/2767-9764.CRC-24-0101

Figure Lengend Snippet: YAP induces Netrin and UNC5 family members in YAP off cancers. A–F, UNC5B Western blots from YAP off cells ectopically expressing YAP or YAP mutants ( A , C , E , F ) or a TEAD4 DNA-binding-domain (DBD)-VP64 fusion protein or controls ( B , D ). Cell lines and tumor types are indicated in each. n = 3. G, UNC5B Western blot from YAP off and YAP on cell lines. n = 2. H, Heatmap showing the effect of ectopic YAP on the expression of UNC5A-D or Netrins (NTN1, 2 and 4) in YAP off cells. * FDR < 0.05; N = not detected. RNA expression levels mined from RNA-Seq data in .

Article Snippet: The following antibodies were used: YAP/TAZ (Santa Cruz Biotech, sc-101199; RRID: AB_1131430), GFP (Santa Cruz Biotech, sc-9996; RRID: AB_627695), TUBULIN (Santa Cruz Biotech, sc-32293; RRID: AB_628412), TEAD1 (BD Biosciences, 610923; RRID: AB_398238), and UNC5B (Cell Signaling Technology, 13851; RRID:AB_2798330).

Techniques: Western Blot, Expressing, Binding Assay, RNA Expression, RNA Sequencing

A Netrin-UNC5-ITGAV/B5 pathway mediates YAP-induced cytostasis. A–D, A netrin blocking antibody (α-NTN) or trapping reagent (UNC5-Fc) alleviates YAP-induced cytostasis in YAP off cell lines. Cell lines and tumor types are indicated in each. Y79 cells expressed wild type YAP, whereas WERI-RB1, NCI-H209 and NCI-H2171 expressed YAP 5SA . *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with untreated (untr) cells; n = 3–5 ( A and B ) or 3 ( C and D ). E, Rescue of YAP-induced cytostasis in control or UNC5B knockout Y79 cells treated with an Integrin-αV/β5 blocking antibody. *, P < 0.05 compared with untreated sgControl cells; n = 3. F, Summary of the mechanism of YAP-induced cytostasis in YAP off cancers.

Journal: Cancer Research Communications

Article Title: Netrin-1 and UNC5B Cooperate with Integrins to Mediate YAP-Driven Cytostasis

doi: 10.1158/2767-9764.CRC-24-0101

Figure Lengend Snippet: A Netrin-UNC5-ITGAV/B5 pathway mediates YAP-induced cytostasis. A–D, A netrin blocking antibody (α-NTN) or trapping reagent (UNC5-Fc) alleviates YAP-induced cytostasis in YAP off cell lines. Cell lines and tumor types are indicated in each. Y79 cells expressed wild type YAP, whereas WERI-RB1, NCI-H209 and NCI-H2171 expressed YAP 5SA . *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with untreated (untr) cells; n = 3–5 ( A and B ) or 3 ( C and D ). E, Rescue of YAP-induced cytostasis in control or UNC5B knockout Y79 cells treated with an Integrin-αV/β5 blocking antibody. *, P < 0.05 compared with untreated sgControl cells; n = 3. F, Summary of the mechanism of YAP-induced cytostasis in YAP off cancers.

Article Snippet: The following antibodies were used: YAP/TAZ (Santa Cruz Biotech, sc-101199; RRID: AB_1131430), GFP (Santa Cruz Biotech, sc-9996; RRID: AB_627695), TUBULIN (Santa Cruz Biotech, sc-32293; RRID: AB_628412), TEAD1 (BD Biosciences, 610923; RRID: AB_398238), and UNC5B (Cell Signaling Technology, 13851; RRID:AB_2798330).

Techniques: Blocking Assay, Control, Knock-Out

Primer sequences used for real-time PCR.

Journal: Frontiers in Oncology

Article Title: Use of long non-coding RNAs for the molecular diagnosis of papillary thyroid cancer

doi: 10.3389/fonc.2022.924409

Figure Lengend Snippet: Primer sequences used for real-time PCR.

Article Snippet: For the analysis of FNA samples, the following TaqMan Assay Mixes were used: LRRC52-AS1 (Hs01594821_m1; JUN-QSY), LINC02082 (Hs00415625_m1; FAM-MGB), UNC5B-AS1 (Hs04274416_g1; ABY-QSY), and GAPDH (Hs02786624_g1; VIC-MGB).

Techniques:

Selected candidate long non-coding RNAs (lncRNAs) from The Atlas of Noncoding RNAs in Cancer (TANRIC) thyroid cancer dataset.

Journal: Frontiers in Oncology

Article Title: Use of long non-coding RNAs for the molecular diagnosis of papillary thyroid cancer

doi: 10.3389/fonc.2022.924409

Figure Lengend Snippet: Selected candidate long non-coding RNAs (lncRNAs) from The Atlas of Noncoding RNAs in Cancer (TANRIC) thyroid cancer dataset.

Article Snippet: For the analysis of FNA samples, the following TaqMan Assay Mixes were used: LRRC52-AS1 (Hs01594821_m1; JUN-QSY), LINC02082 (Hs00415625_m1; FAM-MGB), UNC5B-AS1 (Hs04274416_g1; ABY-QSY), and GAPDH (Hs02786624_g1; VIC-MGB).

Techniques:

Expression levels of candidate long non-coding RNAs (lncRNAs) showing increased expression in surgical specimens at our institution. (A) LRRC52-AS1, (B) LINC02471, (C) LINC02082, (D) UNC5B-AS1, and (E) LINC02408. *p < 0.05, **p < 0.01, and ***p < 0.001.

Journal: Frontiers in Oncology

Article Title: Use of long non-coding RNAs for the molecular diagnosis of papillary thyroid cancer

doi: 10.3389/fonc.2022.924409

Figure Lengend Snippet: Expression levels of candidate long non-coding RNAs (lncRNAs) showing increased expression in surgical specimens at our institution. (A) LRRC52-AS1, (B) LINC02471, (C) LINC02082, (D) UNC5B-AS1, and (E) LINC02408. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: For the analysis of FNA samples, the following TaqMan Assay Mixes were used: LRRC52-AS1 (Hs01594821_m1; JUN-QSY), LINC02082 (Hs00415625_m1; FAM-MGB), UNC5B-AS1 (Hs04274416_g1; ABY-QSY), and GAPDH (Hs02786624_g1; VIC-MGB).

Techniques: Expressing

Expression levels of candidate long non-coding RNAs (lncRNAs) showing decreased expression in surgical specimens at our institution. (A) MPPED2-AS1, (B) LNCNEF, (C) LOC642484, (D) ATP6V0E2-AS1, and (E) LOC100129129. *p < 0.05 and **p < 0.01.

Journal: Frontiers in Oncology

Article Title: Use of long non-coding RNAs for the molecular diagnosis of papillary thyroid cancer

doi: 10.3389/fonc.2022.924409

Figure Lengend Snippet: Expression levels of candidate long non-coding RNAs (lncRNAs) showing decreased expression in surgical specimens at our institution. (A) MPPED2-AS1, (B) LNCNEF, (C) LOC642484, (D) ATP6V0E2-AS1, and (E) LOC100129129. *p < 0.05 and **p < 0.01.

Article Snippet: For the analysis of FNA samples, the following TaqMan Assay Mixes were used: LRRC52-AS1 (Hs01594821_m1; JUN-QSY), LINC02082 (Hs00415625_m1; FAM-MGB), UNC5B-AS1 (Hs04274416_g1; ABY-QSY), and GAPDH (Hs02786624_g1; VIC-MGB).

Techniques: Expressing

Receiver operating characteristic (ROC) curves of candidate long non-coding RNAs (lncRNAs) in The Atlas of Noncoding RNAs in Cancer (TANRIC) thyroid cancer dataset. (A) LRRC52-AS1, (B) LINC02082, (C) UNC5B-AS1, (D) MPPED2-AS1, (E) LNCNEF, (F) LOC100129129. AUC, area under the receiver operating characteristic (ROC) curve.

Journal: Frontiers in Oncology

Article Title: Use of long non-coding RNAs for the molecular diagnosis of papillary thyroid cancer

doi: 10.3389/fonc.2022.924409

Figure Lengend Snippet: Receiver operating characteristic (ROC) curves of candidate long non-coding RNAs (lncRNAs) in The Atlas of Noncoding RNAs in Cancer (TANRIC) thyroid cancer dataset. (A) LRRC52-AS1, (B) LINC02082, (C) UNC5B-AS1, (D) MPPED2-AS1, (E) LNCNEF, (F) LOC100129129. AUC, area under the receiver operating characteristic (ROC) curve.

Article Snippet: For the analysis of FNA samples, the following TaqMan Assay Mixes were used: LRRC52-AS1 (Hs01594821_m1; JUN-QSY), LINC02082 (Hs00415625_m1; FAM-MGB), UNC5B-AS1 (Hs04274416_g1; ABY-QSY), and GAPDH (Hs02786624_g1; VIC-MGB).

Techniques:

Receiver operating characteristic (ROC) curves of long non-coding RNA (lncRNA) combinations (calculated by dividing the increased lncRNA level by the decreased lncRNA level) in The Atlas of Noncoding RNAs in Cancer (TANRIC) thyroid cancer dataset. (A) LRRC52-AS1/MPPED2-AS1, (B) LINC02082/MPPED2-AS1, (C) UNC5B-AS1/MPPED2-AS1, (D) LRRC52-AS1/LNCNEF, (E) LINC02082/LNCNEF, (F) UNC5B-AS1/LNCNEF, (G) LRRC52-AS1/LOC100129129, (H) LINC02082/LOC100129129, and (I) UNC5B-AS1/LOC100129129. AUC, area under the ROC curve.

Journal: Frontiers in Oncology

Article Title: Use of long non-coding RNAs for the molecular diagnosis of papillary thyroid cancer

doi: 10.3389/fonc.2022.924409

Figure Lengend Snippet: Receiver operating characteristic (ROC) curves of long non-coding RNA (lncRNA) combinations (calculated by dividing the increased lncRNA level by the decreased lncRNA level) in The Atlas of Noncoding RNAs in Cancer (TANRIC) thyroid cancer dataset. (A) LRRC52-AS1/MPPED2-AS1, (B) LINC02082/MPPED2-AS1, (C) UNC5B-AS1/MPPED2-AS1, (D) LRRC52-AS1/LNCNEF, (E) LINC02082/LNCNEF, (F) UNC5B-AS1/LNCNEF, (G) LRRC52-AS1/LOC100129129, (H) LINC02082/LOC100129129, and (I) UNC5B-AS1/LOC100129129. AUC, area under the ROC curve.

Article Snippet: For the analysis of FNA samples, the following TaqMan Assay Mixes were used: LRRC52-AS1 (Hs01594821_m1; JUN-QSY), LINC02082 (Hs00415625_m1; FAM-MGB), UNC5B-AS1 (Hs04274416_g1; ABY-QSY), and GAPDH (Hs02786624_g1; VIC-MGB).

Techniques:

Expression levels of candidate long non-coding RNAs (lncRNAs) showing increased expression in fine needle aspiration (FNA) samples at our institution. (A) LRRC52-AS1, (B) LINC02082, and (C) UNC5B-AS1. ***p < 0.001.

Journal: Frontiers in Oncology

Article Title: Use of long non-coding RNAs for the molecular diagnosis of papillary thyroid cancer

doi: 10.3389/fonc.2022.924409

Figure Lengend Snippet: Expression levels of candidate long non-coding RNAs (lncRNAs) showing increased expression in fine needle aspiration (FNA) samples at our institution. (A) LRRC52-AS1, (B) LINC02082, and (C) UNC5B-AS1. ***p < 0.001.

Article Snippet: For the analysis of FNA samples, the following TaqMan Assay Mixes were used: LRRC52-AS1 (Hs01594821_m1; JUN-QSY), LINC02082 (Hs00415625_m1; FAM-MGB), UNC5B-AS1 (Hs04274416_g1; ABY-QSY), and GAPDH (Hs02786624_g1; VIC-MGB).

Techniques: Expressing

Diagnostic performance of long non-coding RNA (lncRNA) expression for the comparison of Bethesda II (n = 23) vs. Bethesda V/VI (n = 18) fine needle aspiration (FNA) samples at our institution.

Journal: Frontiers in Oncology

Article Title: Use of long non-coding RNAs for the molecular diagnosis of papillary thyroid cancer

doi: 10.3389/fonc.2022.924409

Figure Lengend Snippet: Diagnostic performance of long non-coding RNA (lncRNA) expression for the comparison of Bethesda II (n = 23) vs. Bethesda V/VI (n = 18) fine needle aspiration (FNA) samples at our institution.

Article Snippet: For the analysis of FNA samples, the following TaqMan Assay Mixes were used: LRRC52-AS1 (Hs01594821_m1; JUN-QSY), LINC02082 (Hs00415625_m1; FAM-MGB), UNC5B-AS1 (Hs04274416_g1; ABY-QSY), and GAPDH (Hs02786624_g1; VIC-MGB).

Techniques: Diagnostic Assay, Expressing, Comparison

Follow-up results of 10 patients with a thyroid nodule classified as Category III (0 = under the cut-off value, 1 = above the cut-off value).

Journal: Frontiers in Oncology

Article Title: Use of long non-coding RNAs for the molecular diagnosis of papillary thyroid cancer

doi: 10.3389/fonc.2022.924409

Figure Lengend Snippet: Follow-up results of 10 patients with a thyroid nodule classified as Category III (0 = under the cut-off value, 1 = above the cut-off value).

Article Snippet: For the analysis of FNA samples, the following TaqMan Assay Mixes were used: LRRC52-AS1 (Hs01594821_m1; JUN-QSY), LINC02082 (Hs00415625_m1; FAM-MGB), UNC5B-AS1 (Hs04274416_g1; ABY-QSY), and GAPDH (Hs02786624_g1; VIC-MGB).

Techniques:

( A, B ) ChIP-seq profiles of the ( A ) Robo3 and ( B ) Unc5b loci for DNTTIP1 in WT, Dnttip1 KO1 and Elmsan1 KO1 mESCs and for HDAC1 in WT mESCs. Promoter regions used for manual ChIP experiments in ( C ) and ( E ) are highlighted by orange boxes. DNTTIP1 ChIP-seq peaks were determined based on the average of two replicates each from WT mESCs and based on the average of two replicates each from the Dnttip1 KO1 and Elmsan1 KO1 clone, respectively. HDAC1 ChIP-seq data were obtained from GSE55437. ( C ) qPCR from manual ChIP experiments against DNTTIP1, ELMSAN1 and HDAC1 from WT, Dnttip1 KO1 and Elmsan1 KO1 NE targeting the promoter of the Robo3 locus as highlighted in ( A ). IgG was used as a control antibody. ( D ) qRT-PCR for Robo3 mRNA in WT, Dnttip1 KO1 and Elmsan1 KO1 NE after 12 days of differentiation. Expression was normalized to Gapdh. ( E ) qPCR from manual ChIP experiments against DNTTIP1, ELMSAN1 and HDAC1 from WT, Dnttip1 KO1 and Elmsan1 KO1 NE targeting the promoter of the Unc5b locus as highlighted in ( B ). IgG was used as a control antibody. ( F ) qRT-PCR for Unc5b mRNA in WT, Dnttip1 KO1 and Elmsan1 KO1 NE after 12 days of differentiation. Expression was normalized to Gapdh .

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, B ) ChIP-seq profiles of the ( A ) Robo3 and ( B ) Unc5b loci for DNTTIP1 in WT, Dnttip1 KO1 and Elmsan1 KO1 mESCs and for HDAC1 in WT mESCs. Promoter regions used for manual ChIP experiments in ( C ) and ( E ) are highlighted by orange boxes. DNTTIP1 ChIP-seq peaks were determined based on the average of two replicates each from WT mESCs and based on the average of two replicates each from the Dnttip1 KO1 and Elmsan1 KO1 clone, respectively. HDAC1 ChIP-seq data were obtained from GSE55437. ( C ) qPCR from manual ChIP experiments against DNTTIP1, ELMSAN1 and HDAC1 from WT, Dnttip1 KO1 and Elmsan1 KO1 NE targeting the promoter of the Robo3 locus as highlighted in ( A ). IgG was used as a control antibody. ( D ) qRT-PCR for Robo3 mRNA in WT, Dnttip1 KO1 and Elmsan1 KO1 NE after 12 days of differentiation. Expression was normalized to Gapdh. ( E ) qPCR from manual ChIP experiments against DNTTIP1, ELMSAN1 and HDAC1 from WT, Dnttip1 KO1 and Elmsan1 KO1 NE targeting the promoter of the Unc5b locus as highlighted in ( B ). IgG was used as a control antibody. ( F ) qRT-PCR for Unc5b mRNA in WT, Dnttip1 KO1 and Elmsan1 KO1 NE after 12 days of differentiation. Expression was normalized to Gapdh .

Article Snippet: The rescued neurite outgrowth defects of Dnttip1 KO neurons via SLIT3, NTN1 or SLIT3/NTN1 supplementation were blocked by using antibodies directed against the extracellular domain of the axon guidance receptors ROBO3 (R&D Systems, AF3155), UNC5B (R&D Systems, MAB1006), a combination of ROBO3/UNC5B or IgG (Millipore, 12–370) as a negative control.

Techniques: ChIP-sequencing, Control, Quantitative RT-PCR, Expressing

( 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.

Article Snippet: The rescued neurite outgrowth defects of Dnttip1 KO neurons via SLIT3, NTN1 or SLIT3/NTN1 supplementation were blocked by using antibodies directed against the extracellular domain of the axon guidance receptors ROBO3 (R&D Systems, AF3155), UNC5B (R&D Systems, MAB1006), a combination of ROBO3/UNC5B or IgG (Millipore, 12–370) as a negative control.

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.

Article Snippet: The rescued neurite outgrowth defects of Dnttip1 KO neurons via SLIT3, NTN1 or SLIT3/NTN1 supplementation were blocked by using antibodies directed against the extracellular domain of the axon guidance receptors ROBO3 (R&D Systems, AF3155), UNC5B (R&D Systems, MAB1006), a combination of ROBO3/UNC5B or IgG (Millipore, 12–370) as a negative control.

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.

Article Snippet: The rescued neurite outgrowth defects of Dnttip1 KO neurons via SLIT3, NTN1 or SLIT3/NTN1 supplementation were blocked by using antibodies directed against the extracellular domain of the axon guidance receptors ROBO3 (R&D Systems, AF3155), UNC5B (R&D Systems, MAB1006), a combination of ROBO3/UNC5B or IgG (Millipore, 12–370) as a negative control.

Techniques: Activation Assay, Gene Expression, Binding Assay

(A and B) Immunofluorescence staining for Netrin-4 (A) and Unc5B (B) in Vc of the ipsilateral side and contralateral side. Bar:100μm. (C and D) Netrin-4 (green) and NeuN (red) double immunofluorescence staining in Vc of the ipsilateral side. Bar: (C) 100μm (D) 10μm. (E and F) Unc5B (green) and NeuN (red) double immunofluorescence staining in Vc of the ipsilateral side. Bar: (E) 100μm (F) 10μm. (G and H) Netrin-4 (G) and Unc5B (F) mRNA expression in the ipsilateral (ipsi) and contralateral (contra) side at 14days after CCI-Sham injury (n = 3–5). The right graphs show the other day time results. The data are presented as the mean ± SEM. *, P<0.05; Mann-Whitney test.

Journal: PLoS ONE

Article Title: Increased expression of Netrin-4 is associated with allodynia in a trigeminal neuropathic pain model rats by infraorbital nerve injury

doi: 10.1371/journal.pone.0251013

Figure Lengend Snippet: (A and B) Immunofluorescence staining for Netrin-4 (A) and Unc5B (B) in Vc of the ipsilateral side and contralateral side. Bar:100μm. (C and D) Netrin-4 (green) and NeuN (red) double immunofluorescence staining in Vc of the ipsilateral side. Bar: (C) 100μm (D) 10μm. (E and F) Unc5B (green) and NeuN (red) double immunofluorescence staining in Vc of the ipsilateral side. Bar: (E) 100μm (F) 10μm. (G and H) Netrin-4 (G) and Unc5B (F) mRNA expression in the ipsilateral (ipsi) and contralateral (contra) side at 14days after CCI-Sham injury (n = 3–5). The right graphs show the other day time results. The data are presented as the mean ± SEM. *, P<0.05; Mann-Whitney test.

Article Snippet: The following Taqman Assays were used: Netrin-4 (Rn01760394_m1); Unc5B (Rn00573551_m1).

Techniques: Immunofluorescence, Staining, Double Immunofluorescence Staining, Expressing, MANN-WHITNEY

(A and B) Netrin-4 expression in Vc of ipsilateral and contralateral side at 0 and 14days. (C and D) Netrin-4 expression in Vc of ipsilateral and contralateral side after anti-Netrin-4 antibody or Control antibody treatment. (E and F) Unc5B expression in Vc of ipsilateral and contralateral side at 0 and 14days. (G and H) Unc5B expression in Vc of ipsilateral and contralateral side after anti-Netrin-4 antibody or Control antibody treatment. Bars: 100μm.

Journal: PLoS ONE

Article Title: Increased expression of Netrin-4 is associated with allodynia in a trigeminal neuropathic pain model rats by infraorbital nerve injury

doi: 10.1371/journal.pone.0251013

Figure Lengend Snippet: (A and B) Netrin-4 expression in Vc of ipsilateral and contralateral side at 0 and 14days. (C and D) Netrin-4 expression in Vc of ipsilateral and contralateral side after anti-Netrin-4 antibody or Control antibody treatment. (E and F) Unc5B expression in Vc of ipsilateral and contralateral side at 0 and 14days. (G and H) Unc5B expression in Vc of ipsilateral and contralateral side after anti-Netrin-4 antibody or Control antibody treatment. Bars: 100μm.

Article Snippet: The following Taqman Assays were used: Netrin-4 (Rn01760394_m1); Unc5B (Rn00573551_m1).

Techniques: Expressing, Control

UNC5B mediates FLRT2-promoted THP-1 cell maturation into macrophages. FLRT2 was overexpressed by transfecting the hFLRT2 vector, and UNC5B was silenced by transfecting UNC5B-specific shRNA into THP-1 cells. (A) Representative phase-contrast light microscopy images showing morphological alterations of THP-1 cells transfected as indicated (n = 3). Scale bar, 50 μm. (B) qPCR analysis of Itgam , Csf1r , Cd36 , Msr1 , and Olr1 mRNA in THP-1 cells transfected as indicated (n = 3). (C) Immunoblot analysis of CD36, SR-A, UNC5B, and GFP-FLRT2 proteins in THP-1 cells transfected as indicated (n = 3). (D) HUVECs expressing RFP were co-cultured with THP-1 cells transfected as indicated. After 6 h co-culture, pictures were taken using a fluorescence microscope (n = 3). Scale bar, 5 mm. (E) Transwell cell migration assay was performed in THP-1 cells transfected as indicated, and the numbers of the migrated cells were counted (n = 3). Scale bar, 100 μm. (F) Phagocytosis assays were performed by culturing the cells transfected as indicated in Texas red-conjugated zymosan particles for 2 h at 37°C (n = 3). Cells were observed for internalization of the particles by fluorescence microscopy. Scale bar, 50 μm. Data are means ± SD. P values were determined using unpaired, two-tailed Student’s t -tests. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Journal: Frontiers in Immunology

Article Title: Fibronectin leucine-rich transmembrane protein 2 drives monocyte differentiation into macrophages via the UNC5B-Akt/mTOR axis

doi: 10.3389/fimmu.2023.1162004

Figure Lengend Snippet: UNC5B mediates FLRT2-promoted THP-1 cell maturation into macrophages. FLRT2 was overexpressed by transfecting the hFLRT2 vector, and UNC5B was silenced by transfecting UNC5B-specific shRNA into THP-1 cells. (A) Representative phase-contrast light microscopy images showing morphological alterations of THP-1 cells transfected as indicated (n = 3). Scale bar, 50 μm. (B) qPCR analysis of Itgam , Csf1r , Cd36 , Msr1 , and Olr1 mRNA in THP-1 cells transfected as indicated (n = 3). (C) Immunoblot analysis of CD36, SR-A, UNC5B, and GFP-FLRT2 proteins in THP-1 cells transfected as indicated (n = 3). (D) HUVECs expressing RFP were co-cultured with THP-1 cells transfected as indicated. After 6 h co-culture, pictures were taken using a fluorescence microscope (n = 3). Scale bar, 5 mm. (E) Transwell cell migration assay was performed in THP-1 cells transfected as indicated, and the numbers of the migrated cells were counted (n = 3). Scale bar, 100 μm. (F) Phagocytosis assays were performed by culturing the cells transfected as indicated in Texas red-conjugated zymosan particles for 2 h at 37°C (n = 3). Cells were observed for internalization of the particles by fluorescence microscopy. Scale bar, 50 μm. Data are means ± SD. P values were determined using unpaired, two-tailed Student’s t -tests. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Human UNC5B cDNA (HG13606-G) was purchased from Sino Biological and cloned into the BamHI site of the pKmyc vector.

Techniques: Plasmid Preparation, shRNA, Light Microscopy, Transfection, Western Blot, Expressing, Cell Culture, Co-Culture Assay, Fluorescence, Microscopy, Cell Migration Assay, Two Tailed Test

The direct interaction of the FLRT2 ECD with UNC5B is required for FLRT2-promoted THP-1 cell differentiation into macrophages. (A) Myc-tagged hUNC5B and/or Flag-tagged hFLRT2 were transfected into HEK293T cells. Immunoprecipitation samples were collected using anti-Flag antibody, followed by immunoblotting for Myc and Flag (n = 3). (B) Immunolabeling of FLRT2 and UNC5B in PMA-treated THP-1 cells showing the colocalization of endogenous UNC5B and FLRT2. Scale bar, 10 μm. (C) UNC5B was immunoprecipitated from PMA-incubated THP-1 cells, followed by immunoblotting for FLRT2 and UNC5B (n = 3). (D) Schematic illustration of FLRT2 and UNC5B constructs. LRRNT, leucine-rich repeat N-terminal domain. LRR, leucine-rich repeat. LRRCT, leucine-rich repeat C-terminal domain. FNIII, fibronectin type III domain. (E) UNC5B interacts with hFLRT2-ECD but not hFLRT2-ICD (n = 3). (F, G) THP-1 cells were transfected with control, human FLRT2 extracellular domain (hFLRT2-ECD) or human FLRT2 intracellular domain (hFLRT2-ICD) vector for 48 h, followed by qPCR (F) and immunoblot (G) analyses. Data are means ± SD. P values were determined using unpaired, two-tailed Student’s t -tests. NS, not significant. ** P < 0.01, **** P < 0.0001.

Journal: Frontiers in Immunology

Article Title: Fibronectin leucine-rich transmembrane protein 2 drives monocyte differentiation into macrophages via the UNC5B-Akt/mTOR axis

doi: 10.3389/fimmu.2023.1162004

Figure Lengend Snippet: The direct interaction of the FLRT2 ECD with UNC5B is required for FLRT2-promoted THP-1 cell differentiation into macrophages. (A) Myc-tagged hUNC5B and/or Flag-tagged hFLRT2 were transfected into HEK293T cells. Immunoprecipitation samples were collected using anti-Flag antibody, followed by immunoblotting for Myc and Flag (n = 3). (B) Immunolabeling of FLRT2 and UNC5B in PMA-treated THP-1 cells showing the colocalization of endogenous UNC5B and FLRT2. Scale bar, 10 μm. (C) UNC5B was immunoprecipitated from PMA-incubated THP-1 cells, followed by immunoblotting for FLRT2 and UNC5B (n = 3). (D) Schematic illustration of FLRT2 and UNC5B constructs. LRRNT, leucine-rich repeat N-terminal domain. LRR, leucine-rich repeat. LRRCT, leucine-rich repeat C-terminal domain. FNIII, fibronectin type III domain. (E) UNC5B interacts with hFLRT2-ECD but not hFLRT2-ICD (n = 3). (F, G) THP-1 cells were transfected with control, human FLRT2 extracellular domain (hFLRT2-ECD) or human FLRT2 intracellular domain (hFLRT2-ICD) vector for 48 h, followed by qPCR (F) and immunoblot (G) analyses. Data are means ± SD. P values were determined using unpaired, two-tailed Student’s t -tests. NS, not significant. ** P < 0.01, **** P < 0.0001.

Article Snippet: Human UNC5B cDNA (HG13606-G) was purchased from Sino Biological and cloned into the BamHI site of the pKmyc vector.

Techniques: Cell Differentiation, Transfection, Immunoprecipitation, Western Blot, Immunolabeling, Incubation, Construct, Control, Plasmid Preparation, Two Tailed Test

FLRT2 activates Akt/mTOR signaling via the binding of UNC5B with Rac1. (A) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the upregulated differentially expressed genes (DEGs) in PMs of Flrt2 fl/fl and Flrt2 ΔMyel mice. (B) Immunoblot analysis of p-Akt (Ser473), p-Akt (Thr308), Akt, p-mTOR (Ser2481), mTOR, p-4E-BP-1 (Thr37/46), 4E-BP-1, p-S6K (Thr389), S6K, p-S6 (Ser240/244), and S6 proteins in PMs isolated from Flrt2 fl/fl and Flrt2 ΔMyel mice i.p. injected with thioglycollate for 3 days (n = 3 mice per group). (C) Immunoblot analysis of p-Akt (Ser473), p-Akt (Thr308), Akt, p-mTOR (Ser2481), mTOR, p-4E-BP-1 (Thr37/46), 4E-BP-1, p-S6K (Thr389), S6K, p-S6 (Ser240/244), S6, Flag-FLRT2, and FLRT2 protein levels in THP-1 cells transfected with control or hFLRT2 vector for 48 h (n = 3). (D) Immunoblot analysis of the indicated protein levels in THP-1 cells transfected with shNC or shFLRT2 #3 vector for 48 h (n = 3). (E) Immunoprecipitation samples were collected using anti-Flag antibody from control vector and Flag-UNC5B-overexpressing HEK293T cells and subjected to mass spectrometry (MS) to identify potential binding partners of UNC5B protein. A unique peptide of Rac1 protein was identified in the UNC5B-overexpressing immunoprecipitation samples by analyzing the mass-to-charge ratio of the samples. (F) The endogenous interaction between Rac1 and UNC5B was confirmed in THP-1 cells by co-immunoprecipitation (co-IP) analysis. Data are means ± SD. P values were determined using unpaired, two-tailed Student’s t -tests. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Journal: Frontiers in Immunology

Article Title: Fibronectin leucine-rich transmembrane protein 2 drives monocyte differentiation into macrophages via the UNC5B-Akt/mTOR axis

doi: 10.3389/fimmu.2023.1162004

Figure Lengend Snippet: FLRT2 activates Akt/mTOR signaling via the binding of UNC5B with Rac1. (A) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the upregulated differentially expressed genes (DEGs) in PMs of Flrt2 fl/fl and Flrt2 ΔMyel mice. (B) Immunoblot analysis of p-Akt (Ser473), p-Akt (Thr308), Akt, p-mTOR (Ser2481), mTOR, p-4E-BP-1 (Thr37/46), 4E-BP-1, p-S6K (Thr389), S6K, p-S6 (Ser240/244), and S6 proteins in PMs isolated from Flrt2 fl/fl and Flrt2 ΔMyel mice i.p. injected with thioglycollate for 3 days (n = 3 mice per group). (C) Immunoblot analysis of p-Akt (Ser473), p-Akt (Thr308), Akt, p-mTOR (Ser2481), mTOR, p-4E-BP-1 (Thr37/46), 4E-BP-1, p-S6K (Thr389), S6K, p-S6 (Ser240/244), S6, Flag-FLRT2, and FLRT2 protein levels in THP-1 cells transfected with control or hFLRT2 vector for 48 h (n = 3). (D) Immunoblot analysis of the indicated protein levels in THP-1 cells transfected with shNC or shFLRT2 #3 vector for 48 h (n = 3). (E) Immunoprecipitation samples were collected using anti-Flag antibody from control vector and Flag-UNC5B-overexpressing HEK293T cells and subjected to mass spectrometry (MS) to identify potential binding partners of UNC5B protein. A unique peptide of Rac1 protein was identified in the UNC5B-overexpressing immunoprecipitation samples by analyzing the mass-to-charge ratio of the samples. (F) The endogenous interaction between Rac1 and UNC5B was confirmed in THP-1 cells by co-immunoprecipitation (co-IP) analysis. Data are means ± SD. P values were determined using unpaired, two-tailed Student’s t -tests. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Human UNC5B cDNA (HG13606-G) was purchased from Sino Biological and cloned into the BamHI site of the pKmyc vector.

Techniques: Binding Assay, Western Blot, Isolation, Injection, Transfection, Control, Plasmid Preparation, Immunoprecipitation, Mass Spectrometry, Co-Immunoprecipitation Assay, Two Tailed Test

Schematic diagram of our major findings. Pro-differentiation factors, such as PMA, M-CSF, and thioglycollate, upregulate Flrt2 expression in monocytes. FLRT2, in turn, accelerates monocyte-to-macrophage differentiation by binding to UNC5B via its ECD and subsequently activating the Akt/mTOR signaling pathway.

Journal: Frontiers in Immunology

Article Title: Fibronectin leucine-rich transmembrane protein 2 drives monocyte differentiation into macrophages via the UNC5B-Akt/mTOR axis

doi: 10.3389/fimmu.2023.1162004

Figure Lengend Snippet: Schematic diagram of our major findings. Pro-differentiation factors, such as PMA, M-CSF, and thioglycollate, upregulate Flrt2 expression in monocytes. FLRT2, in turn, accelerates monocyte-to-macrophage differentiation by binding to UNC5B via its ECD and subsequently activating the Akt/mTOR signaling pathway.

Article Snippet: Human UNC5B cDNA (HG13606-G) was purchased from Sino Biological and cloned into the BamHI site of the pKmyc vector.

Techniques: Expressing, Binding Assay