Journal: Molecular Oncology

Article Title: Secretome and degradome profiling shows that Kallikrein‐related peptidases 4, 5, 6, and 7 induce TGFβ‐1 signaling in ovarian cancer cells

doi: 10.1016/j.molonc.2013.09.003

Figure Lengend Snippet: (a,b) Western blot detection of PEDF and thrombospondin‐1 in cell conditioned medium. 30 μg proteome were used with a 12.5% reducing SDS‐PAGE. (c) Western blot detection of LAP/TGFβ‐1 in cell conditioned medium. 8 μg proteome were used with a 12.5% reducing SDS‐PAGE. The primary antibody was directed against the LAP domain (prodomain). In line with previous reports (Dubois et al., 1995), the ∼55 kDa band is thought to represent proTGFβ‐1 whereas the predominant ∼40 kDa band is thought to represent LAP. The elevated size in comparison to the primary sequence is thought to stem from glycosylation (Brunner et al., 1992; Dubois et al., 1995). (d) Western blot detection of mature TGFβ‐1 (disulfide linked dimer) in cell conditioned medium. 30 μg proteome were used with a 12.5% non‐reducing SDS‐PAGE. The primary antibody was directed against the actual TGFβ‐1 domain. In addition, N‐terminal degradomics showed TGFβ‐1 activation (see corresponding section). (e) Western blot detection of L1CAM in cell conditioned medium (CCM) and total cell lysate, including cell surface proteins. 8 μg (CCM) and 12 μg proteome (total cell lysate), respectively, were separated with a 7.5% reducing SDS‐PAGE. The molecular weight of 220 kDa for L1CAM is higher than expected but in line with previous reports (Li and Galileo, 2010).

Article Snippet: Primary antibodies [PEDF (1:1000, R&D Systems, AF1177), L1CAM (1:1000, R&D Systems, AF277); LAP (TGFβ‐1 prodomain) (1:1000, R&D Systems, AF246NA); tubulin (1:1000, Sigma, T6199), TGFβ‐1 (1:200, R&D Systems, AF7555)] were incubated for 18 h at 4 °C.

Techniques: Western Blot, SDS Page, Comparison, Sequencing, Glycoproteomics, Activation Assay, Molecular Weight

Journal: Molecular Oncology

Article Title: Secretome and degradome profiling shows that Kallikrein‐related peptidases 4, 5, 6, and 7 induce TGFβ‐1 signaling in ovarian cancer cells

doi: 10.1016/j.molonc.2013.09.003

Figure Lengend Snippet: Immunohistochemical detection of TGFβ‐1 and L1CAM expression in xenografts derived from mice bearing KLK4–7 co‐transfected OV‐MZ‐6 cell and control tumors. A positive staining for both proteins was detected in the cytoplasm, with a more intense staining upon KLK4–7 expression. Scale bars, 50 μm.

Article Snippet: Primary antibodies [PEDF (1:1000, R&D Systems, AF1177), L1CAM (1:1000, R&D Systems, AF277); LAP (TGFβ‐1 prodomain) (1:1000, R&D Systems, AF246NA); tubulin (1:1000, Sigma, T6199), TGFβ‐1 (1:200, R&D Systems, AF7555)] were incubated for 18 h at 4 °C.

Techniques: Immunohistochemical staining, Expressing, Derivative Assay, Transfection, Control, Staining

Journal: Cancer Science

Article Title: Proximity proteomics identifies cancer cell membrane cis ‐molecular complex as a potential cancer target

doi: 10.1111/cas.14108

Figure Lengend Snippet: CHL1 expression in lung tumors from EML4‐ALK transgenic mice. A, EML4‐ALK transgenic mouse lung cancers ( Arrows ). Two representative tumor formations in the lung (upper panel) and HE staining of cancer tissue (lower panel; indicated as the dotted area of “T”). Scale bar: 100 μm. B, RT‐PCR analyses of Gjb4 , MMP13 , CHL1 , Claudin2 and EML4‐ALK mRNA show potent expression in lung cancer tissue. Tissues derived from 12 and 24‐wk old male and female mice were used for the analysis, respectively. N, normal tissue; T, tumor tissue. C, Immunohistochemical staining of lung tissues from EML4‐ALK transgenic mouse. CHL1 staining (upper panel) and Claudin2 staining (lower panel) were performed using anti–CHL1 and anti–Claudin2 antibodies with DIC images. The dotted area indicates the tumor tissue ( T ). D, Protein expression of CHL1 in cancer tissue. Tissue lysate from lung cancer tissue and normal tissue were subjected to western blot analysis using mouse CHL1 antibody. N, normal tissue; T, tumor tissue

Article Snippet: Briefly, EML4‐ALK primary cells, LK2 cells, HEK293 cells and CHL1 transfectant HEK293 cells were washed once with PBS at room temperature and then treated with either 5 μg/mL of HRP‐conjugated anti–mouse CHL1 antibody (AF2147; R&D systems) and anti–human CHL1 antibody (MAB2126; R&D systems) or 4 μg/mL of HRP‐conjugated CTxB (LIST Biological Laboratories) in PBS at room temperature for 20 minutes.

Techniques: Expressing, Transgenic Assay, Staining, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Immunohistochemical staining, Western Blot

Journal: Cancer Science

Article Title: Proximity proteomics identifies cancer cell membrane cis ‐molecular complex as a potential cancer target

doi: 10.1111/cas.14108

Figure Lengend Snippet: BiCAT analysis for cultured cancer cells. A, Representative image of EML4‐ALK primary cells. B, C, Partner molecules with CHL1 in EML4‐ALK primary cells were labeled with fluorescein‐arylazide (B) and fluorescein‐tyramide (C) reagent. Enzyme‐mediated activation of radical source (EMARS) products were, respectively, subjected to western blot analysis followed by staining using anti–fluorescein antibody. “CTxB” indicates the positive control sample using CTxB probe, “CHL1” the samples using CHL1 probe, and “(−)” the negative control samples (no probe). D, EMARS products labeled with fluorescein‐tyramide in LK2 cells. Protein expression level of CHL1 in LK2 and RERF cells (left column). EMARS products by CTxB and human CHL1 probes (right column). Abbreviations are the same as in (C). E, Human receptor tyrosine kinase (RTK) antibody array analysis of EMARS products from LK2 cells. EMARS samples were applied to Human RTK antibody array according to the manufacturer's instructions. “CHL1 probe (+)” indicates the sample using CHL1 probe, and “CHL1 probe (−)” the negative control samples (no probe). The proteins corresponding to positive RTK were indicated in the array data. F, Interaction between FGFR3 and α2 integrin in HEK293 cells. HEK293 (mock) and CHL1 transfectant (hCHL1) cells were subjected to western blot analysis with anti–CHL1 antibody (left panel). The EMARS products by HRP‐conjugated anti CHL1 antibody from HEK293 and CHL1 transfectant cells were subjected to 10% SDS‐PAGE gel followed by direct fluorescein detection (middle panel). Immunoprecipitation experiment of fluorescein‐labeled α2 integrin using anti–fluorescein‐Sepharose (right panel). The immunoprecipitation samples and input lysate were subjected to 6% SDS‐PAGE gel followed by the western blot analysis with anti–α2 integrin antibody

Article Snippet: Briefly, EML4‐ALK primary cells, LK2 cells, HEK293 cells and CHL1 transfectant HEK293 cells were washed once with PBS at room temperature and then treated with either 5 μg/mL of HRP‐conjugated anti–mouse CHL1 antibody (AF2147; R&D systems) and anti–human CHL1 antibody (MAB2126; R&D systems) or 4 μg/mL of HRP‐conjugated CTxB (LIST Biological Laboratories) in PBS at room temperature for 20 minutes.

Techniques: Cell Culture, Labeling, Activation Assay, Western Blot, Staining, Positive Control, Negative Control, Expressing, Ab Array, Transfection, SDS Page, Immunoprecipitation

Journal: Cancer Science

Article Title: Proximity proteomics identifies cancer cell membrane cis ‐molecular complex as a potential cancer target

doi: 10.1111/cas.14108

Figure Lengend Snippet: BiCAT located in lung cancer cell membranes and cellular vesicles. A‐C, Morphological observation of BiCAT in LK2 cells using electron microscopy. Cultured LK2 cells were fixed and co–stained with CHL1 (indicated as 10 nm particles) and partner molecules identified in cell membrane. α2 integrin (A), FGFR3 (B) and contactin1 (C) were indicated as 5 nm particles. Arrows indicate the locations of gold particles. Scale bar: 100‐500 nm

Article Snippet: Briefly, EML4‐ALK primary cells, LK2 cells, HEK293 cells and CHL1 transfectant HEK293 cells were washed once with PBS at room temperature and then treated with either 5 μg/mL of HRP‐conjugated anti–mouse CHL1 antibody (AF2147; R&D systems) and anti–human CHL1 antibody (MAB2126; R&D systems) or 4 μg/mL of HRP‐conjugated CTxB (LIST Biological Laboratories) in PBS at room temperature for 20 minutes.

Techniques: Electron Microscopy, Cell Culture, Staining, Membrane

Journal: Cancer Science

Article Title: Proximity proteomics identifies cancer cell membrane cis ‐molecular complex as a potential cancer target

doi: 10.1111/cas.14108

Figure Lengend Snippet: BiCAT located in the pathological specimens from lung cancer patients. A, Representative images of CHL1‐α2 integrin BiCAT‐positive specimens from 55 cases of lung cancer patients. The lung cancer specimens were co–stained with anti–CHL1 antibody (red) and anti–α2 integrin antibody (green), respectively. DAPI solution was used for the nuclear DNA staining. Then, the resulting specimens were observed with confocal microscopy (×5 objective). Both tumor tissues (upper panel) and normal tissue (middle panel) were stained under the same conditions. Representative images at high magnification observation (×20 objective; lower panel) in part of the positive region of BiCAT indicated as the merged area (yellow). B, C, Quantitative analysis of co–expression signals of CHL1‐α2 integrin BiCAT molecule. The co–expression area was quantified using Image J software as described in the Supporting Materials and Methods (Appendix ). B, Tumor slices. C, Normal slices. The quantitative values of the co–expression signals are shown in mean gray value. D, Statistical analysis of mean gray value of CHL1‐α2 integrin BiCAT between normal and tumor tissues. The analysis was performed with the Mann–Whitney test using R software and EZR. P < 1 × 10 −7 . The CHL1‐α2 integrin BiCAT had significantly higher expression in tumor tissues

Article Snippet: Briefly, EML4‐ALK primary cells, LK2 cells, HEK293 cells and CHL1 transfectant HEK293 cells were washed once with PBS at room temperature and then treated with either 5 μg/mL of HRP‐conjugated anti–mouse CHL1 antibody (AF2147; R&D systems) and anti–human CHL1 antibody (MAB2126; R&D systems) or 4 μg/mL of HRP‐conjugated CTxB (LIST Biological Laboratories) in PBS at room temperature for 20 minutes.

Techniques: Staining, Confocal Microscopy, Expressing, Software, MANN-WHITNEY

Journal: Cancer Science

Article Title: Proximity proteomics identifies cancer cell membrane cis ‐molecular complex as a potential cancer target

doi: 10.1111/cas.14108

Figure Lengend Snippet: In vitro simulation of effective drug combination to inhibit cancer cell proliferation based on BiCAT information. A, The single and double administration under daily treatment protocol (n = 5). The administration timing is indicated by closed triangles. The cell numbers of the treated cells were measured on Day 2 and Day 4. B, The relative ratio (% of non–treated cells as control) of cell proliferation rates in LK2 cells and EML4‐ALK primary cells. The statistical analysis was performed using Tukey's test and Dunnett's multiple test. The results from Dunnett's test are presented in Figure 6; * P < 0.05; ** P < 0.005; ***P < 0.001. C, Double administration of molecular targeted reagents leads to changes in the expression of partner molecules. The samples of single and double administration under daily treatment conditions (3 d) in LK2 cells were subjected to phos‐tag SDS‐PAGE and then western blot analysis using CHL1, α2 integrin and FGFR3 antibodies. The CBB staining image indicates load control. The molecular weight markers were not shown in this figure because phos‐tag SDS‐PAGE cannot show the correct molecular weight of sample proteins. D, Western blot analysis of phosphorylated FGFR3 in single and double administration samples. The samples under daily treatment conditions (3 d) in LK2 cells were subjected to normal SDS‐PAGE gel and then western blot analysis using anti–FGFR3 and anti–phospho‐FGFR3 antibodies. The quantification of the phosphorylated bands detected in FGFR3 blots was performed using Image J software (ver. 1.51). The ratio of phosphorylation among the samples is indicated below the figure. E, Western blot analysis of phospho‐focal adhesion kinase (FAK) in single and double administration samples. The samples under daily treatment conditions (3 d) in LK2 cells were subjected to normal SDS‐PAGE and then western blot analysis using anti–FAK and anti–phospho‐FAK antibodies

Article Snippet: Briefly, EML4‐ALK primary cells, LK2 cells, HEK293 cells and CHL1 transfectant HEK293 cells were washed once with PBS at room temperature and then treated with either 5 μg/mL of HRP‐conjugated anti–mouse CHL1 antibody (AF2147; R&D systems) and anti–human CHL1 antibody (MAB2126; R&D systems) or 4 μg/mL of HRP‐conjugated CTxB (LIST Biological Laboratories) in PBS at room temperature for 20 minutes.

Techniques: In Vitro, Control, Expressing, SDS Page, Western Blot, Staining, Molecular Weight, Software, Phospho-proteomics

Journal: Open Medicine

Article Title: CHL1 and NrCAM are Primarily Expressed in Low Grade Pediatric Neuroblastoma

doi: 10.1515/med-2019-0109

Figure Lengend Snippet: Expression of CHL1 and NrCAM in pediatric neuroblastoma. Representative examples of CHL1 positive (A) and CHL-1 negative (B) (magnification x100) as well as NrCAM positive (C) and NrCAM negative (D) immunostaining (magnification x200).

Article Snippet: Afterwards, the primary antibody either specific for CHL1 (goat, polyclonal antibody: AF2126, R&D Systems, MN, USA) or NrCAM (goat anti-human NrCAM antibody: AF2034, R&D Systems, MN, USA,) was applied at 37°C and pH 9.1 for 60 minutes.

Techniques: Expressing, Immunostaining

Journal: Open Medicine

Article Title: CHL1 and NrCAM are Primarily Expressed in Low Grade Pediatric Neuroblastoma

doi: 10.1515/med-2019-0109

Figure Lengend Snippet: Kaplan-Meier survival curves for overall and event-free survival. No association was found for CHL1-expression (A/B). Survival rates were better by trend in children with NrCAM positive tumors (C/D) but without statistical significance (p=0.07 and p=0.06).

Article Snippet: Afterwards, the primary antibody either specific for CHL1 (goat, polyclonal antibody: AF2126, R&D Systems, MN, USA) or NrCAM (goat anti-human NrCAM antibody: AF2034, R&D Systems, MN, USA,) was applied at 37°C and pH 9.1 for 60 minutes.

Techniques: Expressing

Journal: Open Medicine

Article Title: CHL1 and NrCAM are Primarily Expressed in Low Grade Pediatric Neuroblastoma

doi: 10.1515/med-2019-0109

Figure Lengend Snippet: CHL1 expression as well as clinical, pathologic and molecular characteristics of the analysed neuroblastoma tissue samples. Statistical analyses by using cross-tables, two-sided Fisher´s and Chi-squared test.

Article Snippet: Afterwards, the primary antibody either specific for CHL1 (goat, polyclonal antibody: AF2126, R&D Systems, MN, USA) or NrCAM (goat anti-human NrCAM antibody: AF2034, R&D Systems, MN, USA,) was applied at 37°C and pH 9.1 for 60 minutes.

Techniques: Expressing, Amplification