ig1 domain  (Addgene inc)

Journal: Nature Communications

Article Title: Coadaptation fostered by the SLIT2-ROBO1 axis facilitates liver metastasis of pancreatic ductal adenocarcinoma

doi: 10.1038/s41467-023-36521-0

Figure Lengend Snippet: a Representative IF staining showing the location of SLIT2 and CK19 in adjacent liver or liver metastases of PDAC patients. SLIT2, green; CK19, red; DAPI, blue ( n = 35 patients, 3 fields assessed per sample). Scale bar, 100 μm. b Representative IHC-P staining of CK19 and SLIT2 in liver metastatic niches in PDAC patients ( n = 35 cases, 3 fields assessed per sample). Scale bars, 100 μm. c, d Representative IF staining showing the location of SLIT2 and albumin in the PMN in the Kras G12D/+ / Trp53 R172H/+ / Pdx1 -Cre (KPC) mouse model ( c ) or adjacent liver of metastatic niches in PDAC patients ( d ). Albumin, green; SLIT2, red; DAPI, blue ( n = 35 samples for patients, n = 6 samples per group for mouse models, 3 fields assessed per sample). Scale bars, 50 μm. e Representative IF staining showing the location of ROBO1 and CK19 in the liver metastasis of PDAC patients. ROBO1, green; CK19, red; DAPI, blue ( n = 35 samples, 3 fields assessed per sample). Scale bar, 50 μm. f Representative RNAscope staining displaying the location of SLIT2 mRNA and ALB (upper), KRT19 (middle) and ACTA1 (bottom) mRNAs in the liver metastasis (LM) of PDAC patients. ALB , KRT19 or ACTA1 , green; SLIT2, red; DAPI, blue ( n = 5 samples, 3 fields assessed per sample). Scale bar, 100 μm. g Representative RNAscope staining displaying the location of Slit2 mRNA and Alb mRNA in the premetastatic niche (PMN) and macrometastatic niche (MMN) of Kpc1199 model mice. Alb , green; Slit2 , red; DAPI, blue ( n = 5 samples, 3 fields assessed per sample). Scale bar, 100 μm. h Representative IF staining displaying the expression of ROBO1 in metastatic livers with or without SLIT2 enrichment. CK19, green; ROBO1, red; DAPI, blue ( n = 6 mice per group, 3 fields assessed per sample). Scale bar, 500 μm. Source data are provided in the Source Data file.

Article Snippet: A neutralizing antibody against the Ig1-Ig2 domain of ROBO1 (HAM1H6-1-8) and negative control IgG were purchased from HuaBio.

Techniques: Staining, Paraffin-embedded Immunohistochemistry, RNAscope, Expressing

Journal: Nature Communications

Article Title: Coadaptation fostered by the SLIT2-ROBO1 axis facilitates liver metastasis of pancreatic ductal adenocarcinoma

doi: 10.1038/s41467-023-36521-0

Figure Lengend Snippet: a – c Representative CT combined with 3D organ reconstruction bioluminescence imaging to assess liver metastasis modelled by Kpc1199 Robo1 -FL ( b ) or Panc02 Robo1 -FL ( c ) cells in Slit2 fl/fl or Slit2 fl/fl / Alb -Cre mice with or without Lenti- loxp-Slit2 injection: Premetastatic niche (PMN) (upper) and macrometastatic niche (MMN) (lower) ( n = 5 mice per group, mean ± SEM.; two-tailed unpaired Student’s t test). Scale colour bars: 2.00 × 10 5 −2.00 × 10 7 . d, e Representative CT combined with 3D organ reconstruction bioluminescence imaging displaying Kpc1199 Robo1 -FL (upper) and Panc02 Robo1 -FL (lower) cell-injected liver metastasis mouse models administered IgG or ROBO1 neutralizing antibody (n = 5 mice per group, mean ± SEM.; two-tailed unpaired t test). Scale colour bar: 2.00 × 10 5 −2.00 × 10 7 . Red, reconstructed liver; orange, reconstructed spleen; yellow, signals of tumour niches. ( f – h ) The administration of IgG or ROBO1 neutralizing antibody to the Kras G12D/+ / Trp53 R172H/+ / Pdx1 -Cre (KPC) mouse model from week 12 is shown. The number of metastatic niches in the liver with diameters over or under 500 μm in each group was determined ( g ), and the percentage of ROBO1 + metastatic niches in each group was assessed ( h ) ( n = 5 mice per group, mean ± SEM.; two-tailed unpaired t test). ( i ) IHC-P staining of SLIT2 and ROBO1 in serial sections of livers with metastasis from KPC mice with or without ROBO1 neutralizing antibody treatment ( n = 5 mice per group, 3 fields assessed per sample). Scale bar, 200 μm. ( j – l ) IHC-P staining of SLIT2 and ROBO1 in serial sections of livers with metastasis from KPC mice that underwent ROBO1 neutralizing antibody treatment starting from PMN (week 12-) or MMN (week 16-) occurrence. The number of metastatic niches in the liver with diameters over or under 500 μm in each group was determined ( k ), and the percentage of ROBO1 + metastatic niches in each group was assessed ( l ) ( n = 5 mice per group, mean ± SEM.; two-tailed unpaired t test). ( m ) IHC-P staining of SLIT2 in serial sections of KPC liver metastases that experienced ROBO1 neutralizing antibody treatment starting from PMN (week 12) or MMN (week 16) occurrence ( n = 5 mice per group, 3 fields assessed per sample). Scale bar, 200 μm. Source data are provided in the Source Data file.

Article Snippet: A neutralizing antibody against the Ig1-Ig2 domain of ROBO1 (HAM1H6-1-8) and negative control IgG were purchased from HuaBio.

Techniques: Imaging, Injection, Two Tailed Test, Paraffin-embedded Immunohistochemistry, Staining

Journal: Nature Communications

Article Title: Coadaptation fostered by the SLIT2-ROBO1 axis facilitates liver metastasis of pancreatic ductal adenocarcinoma

doi: 10.1038/s41467-023-36521-0

Figure Lengend Snippet: a Representative H&E staining and IHC-P staining of ROBO1 in human PDAC primary tumours and paired liver metastatic niches displaying the importance of ROBO1 in liver metastasis ( n = 35 cases, 3 fields assessed per sample). Scale bars, 100 μm. b The number of PDAC primary tumours or liver metastases expressing different ROBO1 levels according to scores on IHC-P staining is shown ( n = 35 cases). c Equal amounts of Panc02 Ctrl and Panc02 Robo1 -FL/GFP cells were mixed to prepare an intrasplenic injection model followed by metastatic niche separation and reculture. IHC-P and flow cytometry were performed to evaluate coadaptation. PG0: original cell mixture; PG1: cells derived from PG0-modelled liver metastases; PG2: cells derived from PG1-modelled liver metastases. d–e Flow cytometry to detect the cell composition of PG0, PG1 or PG2 ( n = 5 technical repeats per group). f Heatmap showing the distribution of ROBO1 and SLIT2 expression in the livers of Panc02 cell mixture-modelled mice measured by IHC-P staining ( n = 32 mice per group, 3 fields assessed per sample; independent experiments for each group). N: negative; W, weak; M, moderate; P, positive. g, h Mice treated with ROBO1 neutralizing antibody were modelled with a Panc02 cell mixture before IHC-P staining was performed ( n = 32 mice per group, 3 fields assessed per sample; independent experiments for each group). N: negative; W, weak; M, moderate; P, positive. i The strategy for exploring the coadaptation mechanism mediated by SLIT2-ROBO1 in liver metastatic niches. A mixture of equal amounts of Kpc1199 Robo1 -FL/GFP cells and Kpc1199 ΔRobo1/mCherry cells (Mix-I) or Kpc1199 Robo1 -FL/GFP cells and Kpc1199 CTRL /mCherry cells (Mix-II) was intrasplenically injected into Slit2 /CKO or CTRL mice for further examination. j, k Representative IF staining of a Kpc1199 cell mixture showing the formation of liver metastatic niches indicating coadaptation ( n = 5 mice per group, 3 fields assessed per sample, mean ± SEM.; two-tailed unpaired t test). GFP, green; mCherry, red; DAPI, blue. Scale bars, 100 μm. l Flow cytometry detection of the ratio of two types of Kpc1199 cells in separated liver metastatic niches. Source data are provided in the Source Data file.

Article Snippet: A neutralizing antibody against the Ig1-Ig2 domain of ROBO1 (HAM1H6-1-8) and negative control IgG were purchased from HuaBio.

Techniques: Staining, Paraffin-embedded Immunohistochemistry, Expressing, Injection, Flow Cytometry, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Coadaptation fostered by the SLIT2-ROBO1 axis facilitates liver metastasis of pancreatic ductal adenocarcinoma

doi: 10.1038/s41467-023-36521-0

Figure Lengend Snippet: a – c Representative CT combined with 3D organ reconstruction bioluminescence imaging displaying the outgrowth ability of Kpc1199 cells ( b ) or Panc02 cells ( c ) in mouse models: Premetastatic niche (PMN) (upper) and macrometastatic niche (MMN) (lower) ( n = 5 mice per group, mean ± SEM.; two-tailed unpaired t test). Scale colour bars: 2.00 × 10 5 −2.00 × 10 7 . d, e Survival analysis of intrasplenic mouse models bearing Kpc1199 or Panc02 injection ( e ) ( n = 8 mice per group). f Viability of SW-1990 cells with (+rSLIT2) or without (+PBS) 30 nM rSLIT2 exposure ( n = 2 biological replicates, mean ± SEM., one-way repeated-measures ANOVA). The Y-axis represents OD values at 450 nm. ( g ) Viability of SW-1990 ROBO1 -FL cells exposed to 30 nM rSLIT2 with or without ROBO1 neutralizing antibody ( n = 2 biological replicates, mean ± SEM., one-way repeated-measures ANOVA). The Y-axis represents OD values at 450 nm. ( h ) Colony formation assays evaluating the outgrowth ability of SW-1990 cells expressing ROBO1-FL or ΔROBO1 exposed to 10 nM or 30 nM rSLIT2 ( n = 2 biological replicates, mean ± SEM.; two-tailed unpaired t test); ( i ) Tumour sizes in subcutaneous xenograft models utilizing PANC-1 shCTRL or PANC-1 sh ROBO1 cells ( n = 2 biological replicates, n = 7 mice per group, tumour volume was calculated as volume = 0.5 × length × width2, mean ± SEM., one-way Repeat-Measure ANOVA). j Tumour growth in subcutaneous xenograft models utilizing CAPAN-1 CTRL or CAPAN-1 SLIT2- oe cells ( n = 2 biological replicates, n = 6 mice per group, tumour volume was calculated as volume = 0.5 × length × width2, mean ± SEM., one-way repeated-measures ANOVA). k Apoptosis of SW-1990 measured by flow cytometry with dual PI and Annexin V staining ( n = 2 biological replicates, n = 3 tests per group, mean ± SEM.; two-tailed unpaired t test). ( l ) TUNEL assays performed on SW-1990 CTRL , SW-1990 ΔROBO1 and SW-1990 ROBO1 -FL cells with or without 30 nM rSLIT2 exposure ( n = 3 biological replicates, 3 fields assessed per sample, mean ± SEM., two-tailed unpaired t test). TUNEL staining, green; DAPI, blue. Scale bars: 50 μm. ( m ) Caspase-3/7 activity in SW-1990 cells measured 48 h after serum starvation ( n = 3 biological replicates, n = 5 tests per group, mean ± SEM., two-tailed unpaired t test). n Tumour growth in subcutaneous xenograft models utilizing SW-1990 CTRL , SW-1990 ΔROBO1 and SW-1990 ROBO1 -FL cells measured every 4 days ( n = 6 mice per group, tumour volume was calculated as volume = 0.5 × length × width2, mean ± SEM., one-way Repeat-Measure ANOVA). Source data are provided in the Source Data file.

Article Snippet: A neutralizing antibody against the Ig1-Ig2 domain of ROBO1 (HAM1H6-1-8) and negative control IgG were purchased from HuaBio.

Techniques: Imaging, Two Tailed Test, Injection, Expressing, Flow Cytometry, Staining, TUNEL Assay, Activity Assay

Journal: Nature Communications

Article Title: Coadaptation fostered by the SLIT2-ROBO1 axis facilitates liver metastasis of pancreatic ductal adenocarcinoma

doi: 10.1038/s41467-023-36521-0

Figure Lengend Snippet: a Viability of SW-1990 ROBO1 -FL cells exposed to 30 nM rSLIT2 with or without the p38αMAPK-specific inhibitor VX-702 (5 μM) ( n = 2 biological replicates, mean ± SEM., one-way repeated-measures ANOVA). The Y-axis represents OD values at 450 nm. b Viability of SW-1990 CTRL and SW-1990 ROBO1 -FL cells treated with 30 nM rSLIT2 with or without the p38αMAPK-specific inhibitor VX-702 at various concentrations (0.2 μM, 1 μM, 5 μM, 25 μM, 125 μM; time point = D3; n = 2 biological replicates; mean ± SEM.; one-way Repeat-Measure ANOVA). ns. no significant difference, P > 0.05 . The Y-axis represents OD values at 450 nm. c WB displaying the time-dependent rSLIT2-induced p38αMAPK phosphorylation in SW-1990 Robo1 -FL cells ( n = 2 biological replicates). d WB showing the phosphorylation of p38αMAPK in PANC-1, MIA PaCa-2 or SW-1990 cell lines treated with 30 nM rSLIT2 ( n = 2 biological replicates). e WB displaying the relationship of SLIT2 (30 nM), ROBO1 and MEK3/6 expression with phosphorylation of MEK3/6 or p38αMAPK ( n = 2 biological replicates). f Representative ICC staining in 30 nM rSLIT2-treated SW-1990 CTRL , SW-1990 ΔROBO1 and SW-1990 ROBO1 -FL cells displaying the nuclear translocation of p38αMAPK (3 fields assessed per sample). P-p38MAPK, red; DAPI, blue. Scale bars: 50 μm ( n = 2 biological replicates). g Representative ex vivo IHC-P staining of P-p38 showing rSLIT2-induced phosphorylation of p38αMAPK in cultured Panc02 Robo1 -FL cells from metastases in mouse livers ( n = 6 samples per group, 3 fields assessed per sample). Scale bars, 100 μm. ( h ) Representative ex vivo IF staining of P-p38 and ROBO1 in Kras G12D/+ / Trp53 R172H/+ / Pdx1 -Cre (KPC) mouse model metastatic niches formed in livers with or without ROBO1 neutralizing antibody treatment. ROBO1, green; P-p38MAPK, red; DAPI, blue ( n = 3 mice per group, 3 fields assessed per sample). Scale bar, 50 μm. I , j Lysate of the PANC-1 cell line endogenously immunoprecipitated with anti-ROBO1 and anti-p38α MAPK ( i ) or anti-ROBO1 and anti-MEK3/6 ( j ) antibodies and immunoblotted with the indicated antibodies ( n = 2 biological replicates). k Lysate of rSLIT2 (30 nM)-induced SW-1990 cells immunoprecipitated with anti-ROBO1 antibody and immunoblotted with the indicated antibodies ( n = 2 biological replicates). Source data are provided in the Source Data file.

Article Snippet: A neutralizing antibody against the Ig1-Ig2 domain of ROBO1 (HAM1H6-1-8) and negative control IgG were purchased from HuaBio.

Techniques: Phospho-proteomics, Expressing, Staining, Translocation Assay, Ex Vivo, Paraffin-embedded Immunohistochemistry, Cell Culture, Immunoprecipitation

Journal: Nature Communications

Article Title: Coadaptation fostered by the SLIT2-ROBO1 axis facilitates liver metastasis of pancreatic ductal adenocarcinoma

doi: 10.1038/s41467-023-36521-0

Figure Lengend Snippet: ( a ) Summary model displaying the process of SLIT2-ROBO1-mediated coadaptation of hepatic cells and tumour cells, which promotes metastatic niche outgrowth in PDAC. b Summary model showing the dual roles of ROBO1 in coadaptation with or without SLIT2. ROBO1 induces apoptosis through the caspase cascade in the absence of SLIT2 and triggers proliferation and survival through the p38MAPK pathway by interacting with SLIT2.

Article Snippet: A neutralizing antibody against the Ig1-Ig2 domain of ROBO1 (HAM1H6-1-8) and negative control IgG were purchased from HuaBio.

Techniques:

Journal: Nature Communications

Article Title: Coadaptation fostered by the SLIT2-ROBO1 axis facilitates liver metastasis of pancreatic ductal adenocarcinoma

doi: 10.1038/s41467-023-36521-0

Figure Lengend Snippet: Shown are shRNA sequences

Article Snippet: A neutralizing antibody against the Ig1-Ig2 domain of ROBO1 (HAM1H6-1-8) and negative control IgG were purchased from HuaBio.

Techniques: shRNA, Sequencing

Journal: Nature Communications

Article Title: Coadaptation fostered by the SLIT2-ROBO1 axis facilitates liver metastasis of pancreatic ductal adenocarcinoma

doi: 10.1038/s41467-023-36521-0

Figure Lengend Snippet: Shown are primer sequences used in this article

Article Snippet: A neutralizing antibody against the Ig1-Ig2 domain of ROBO1 (HAM1H6-1-8) and negative control IgG were purchased from HuaBio.

Techniques:

Journal: Journal of molecular biology

Article Title: Conformational Change of the Hairpin-like-structured Robo2 Ectodomain Allows NELL1/2 Binding.

doi: 10.1016/j.jmb.2022.167777

Figure Lengend Snippet: Figure 4. FRET-based indicators to assess the topological conformation of the Robo2 ectodomain. (A) Schematic diagrams of the intramolecular FRET constructs. Gamillus/GFP and mTurquoise2/CFP were fused to the N- and C-termini of the Robo2 ectodomain, respectively. A deletion construct of both the Ig1 and FNIII3 domains was prepared to decrease the distance between Gamillus and mTurquoise2. (B) Schematic overview of the FRET experiments. If Gamillus and mTurquoise2 are in close proximity due to conformational change of the Robo2 ectodomain, FRET (emission of 515-nm light after excitation with 430-nm light) may occur. (C) Emission scans of Robo2-ECD and Robo2-ECD/DIg1&FNIII3 constructs at pH 7.0. The excitation wavelength was 430 nm and emission was measured at 460–560 nm. (D) The FRET ratio was defined as the ratio of emission intensity of Gamillus to the emission intensity of mTurquoise2 (515 ± 5 nm/475 ± 5 nm). Each value represents the mean ± SD of at least triplicate results. *, p < 0.05 (Student’s t-test).

Article Snippet: Gamillus/ pcDNA3 was a gift from Takeharu Nagai (Addgene plasmid # 124837).38 pLifeAct- mTurquoise2 was a gift from Dorus Gadella (Addgene plasmid # 36201).37 Deletion mutants lacking the coding sequence for both the Ig1 domain (residues 43–144) and the FNIII3 domain (residues 740–826) or only Ig1 domain were generated by inverse PCR and designated as Gam:Rob o2-ECD/DIg1&FNIII3:mTq2 and Gam:Robo2ECD/DIg1:mTq2, respectively.

Techniques: Construct

Journal: Scientific Reports

Article Title: SALM5 trans-synaptically interacts with LAR-RPTPs in a splicing-dependent manner to regulate synapse development

doi: 10.1038/srep26676

Figure Lengend Snippet: ( a–c ) Coprecipitation between recombinant ectodomains of SALM5 and LAR. LAR ectodomain proteins (LAR-Ecto-Fc or LAR-IgFN14-Fc [Ig1-3 + FN1-4]) and HA-SALM5-Ecto secreted from transfected HEK293T cells into the supernatant were subjected to immunoprecipitation (IP) and immunoblot analysis. ( d–g ) Direct interaction between purified, soluble LAR and SALM5 fusion proteins, measured by the biolayer interferometry. Hybrid SALM5-Ecto proteins binds LAR-Ig-FN12 and LAR-Ig but not to LAR-FN12 fusion proteins ( d–f ), which is summarized in a schematic diagram ( g ). A hybrid SALM5 protein was used to increase protein expression levels (see Materials and methods for details). Note that the LAR constructs longer than the Ig domains only used here (LAR-FN12) and in the coimmunoprecipitation experiments (LAR-Ig-FN14) would not interfere with demonstrating the direction interaction of LAR with SALM5, as evident in .

Article Snippet: The Ig1-3-FNIII12 domain (aa 27A–513A), Ig1-3-A + B + (aa 30D-318L), and FN12 (aa 312Q–510G) of human LAR were cloned into pAcGP67 (BD Bioscience), modified for C-terminal protein-A tagging.

Techniques: Recombinant, Transfection, Immunoprecipitation, Western Blot, Purification, Expressing, Construct