Journal: Cellular oncology (Dordrecht)

Article Title: WNT5A-ROR2 axis mediates VEGF dependence of BRAF mutant melanoma.

doi: 10.1007/s13402-022-00757-7

Figure Lengend Snippet: Fig. 4 BRAFV600E knock-in influences the expression of genes in growth factor and angiogenesis pathways. (A) Expression heatmap of top 50 differentially expressed genes (DEGs) identified in microarray analysis of isogenic BRAFWT and BRAFV600E melanoma clones and parental CHL1 cells. (B) Top 10 signaling pathways represented by DEGs in the BRAFV600E and wildtype clones. Numbers in brackets refer to the numbers of genes annotated within each pathway (http://www.pantherdb.org)

Article Snippet: Human melanoma cell lines CHL1, SKMEL2 and HCMB were obtained from the American Type Culture Collection.

Techniques: Knock-In, Expressing, Microarray, Clone Assay, Protein-Protein interactions

Journal: Oncology Letters

Article Title: Overexpression of close homolog of L1 enhances the chemosensitivity of lung cancer cells via inhibition of the Akt pathway

doi: 10.3892/ol.2020.11972

Figure Lengend Snippet: CHL1 is downregulated in DDP and PTX-resistant A549 cells. (A) Cell survival of A549 and A549-resistant cells (A549/DDP and A549/PTX) treated with increasing concentrations of DDP and PTX, as assessed by MTT assay. (B) The IC 50 values of DDP in A549/DDP and A549 cells, and the IC 50 values of PTX in A549/PTX and A549 cells. *P<0.05 vs. A549 cells. (C) Western blotting demonstrated the expression of drug resistance-related proteins MDR1, MRP and LRP in A549 cells and A549-resistant cells (A549/DDP and A549/PTX). *P<0.05 vs. A549 cells. The protein and mRNA expression levels of CHL1 in A549 cells and A549-resistant cells (A549/DDP and A549/PTX) were analysed by (D) western blotting and (E) reverse transcription-quantitative PCR, respectively. *P<0.05 vs. A549 cells. (F) The mRNA expression of CHL1 in H460 and H460/DDP cells in the GSE21656 dataset. *P<0.05 vs. H460 cells. CHL1, close homolog of L1; DDP, cisplatin; PTX, paclitaxel; MDR1, multi-drug resistance gene 1; MRP, multidrug resistance-associated protein; LRP, low-density lipoprotein receptor-related protein; IC50, half maximal inhibitory concentration.

Article Snippet: The resistant cells A549/PTX and A549/DDP cells were transfected with 4.0 µg CHL1 recombinant expression plasmid (cat. no. HG10143-NY; Sino Biological, Inc.).

Techniques: MTT Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Concentration Assay

Journal: Oncology Letters

Article Title: Overexpression of close homolog of L1 enhances the chemosensitivity of lung cancer cells via inhibition of the Akt pathway

doi: 10.3892/ol.2020.11972

Figure Lengend Snippet: CHL1-knockdown increases A549 cell resistance to DDP and PTX. (A) Western blotting was performed to validate the efficiency of transfection with CHL1 siRNAs. *P<0.05 vs. scramble. MTT assays were performed to determine the survival rate of CHL1-knockdown A549 cells treated with (B) 0–2.5 µg/ml DDP or (C) 0–50 ng/ml DDP. (D) Colony formation assay of A549 cells transfected with CHL1 siRNA in the presence or absence of 1.5 µg/ml DDP or 35 ng/ml PTX. (E) Flow cytometry analysis was used to detect apoptosis in A549 cells transfected with CHL1 siRNA in the presence or absence of 1.5 µg/ml DDP or 35 ng/ml PTX. *P<0.05, ***P<0.001. CHL1, close homolog of L1; DDP, cisplatin; PTX, paclitaxel; si, small interfering.

Article Snippet: The resistant cells A549/PTX and A549/DDP cells were transfected with 4.0 µg CHL1 recombinant expression plasmid (cat. no. HG10143-NY; Sino Biological, Inc.).

Techniques: Western Blot, Transfection, Colony Assay, Flow Cytometry

Journal: Oncology Letters

Article Title: Overexpression of close homolog of L1 enhances the chemosensitivity of lung cancer cells via inhibition of the Akt pathway

doi: 10.3892/ol.2020.11972

Figure Lengend Snippet: Overexpression of CHL1 increases the sensitivity of resistant A549 cells to DDP and PTX. (A) Western blotting was performed to detect CHL1 expression in A549/DDP and A549/PTX cells transfected with CHL1 expression plasmids. *P<0.05 vs. vector. Effect of CHL1 overexpression on resistant A549 cell survival rate when treated with (B) 0–10 µg/ml DDP or (C) 0–250 ng/ml PTX, as determined by MTT assay. (D) Colony formation assays demonstrated the number of colonies of resistant A549 cells transfected with CHL1 expression plasmids in the presence or absence of 8 µg/ml DDP or 160 ng/ml PTX. (E) Flow cytometry analysis was performed to assess apoptosis in resistant A549 cells transfected with CHL1 expression plasmids in the presence or absence of 8 µg/ml DDP or 160 ng/ml PTX. CHL1 overexpression enhanced chemosensitivity of A549/DDP cells to DDP in vivo , which was demonstrated by the effect of DDP treatment or CHL1 overexpression on the (F) growth and (G) weight of xenografts derived from A549/DDP cells. *P<0.05, **P<0.01. CHL1, close homolog of L1; DDP, cisplatin; PTX, paclitaxel.

Article Snippet: The resistant cells A549/PTX and A549/DDP cells were transfected with 4.0 µg CHL1 recombinant expression plasmid (cat. no. HG10143-NY; Sino Biological, Inc.).

Techniques: Over Expression, Western Blot, Expressing, Transfection, Plasmid Preparation, MTT Assay, Flow Cytometry, In Vivo, Derivative Assay

Journal: Oncology Letters

Article Title: Overexpression of close homolog of L1 enhances the chemosensitivity of lung cancer cells via inhibition of the Akt pathway

doi: 10.3892/ol.2020.11972

Figure Lengend Snippet: CHL1 mediates DDP and PTX sensitivity by inhibiting Akt activity. (A) Western blotting was performed to detect the expression of p-Akt and total Akt in CHL-silenced and -restored cell models. *P<0.05 vs. scramble or vector. (B) MTT assays were performed to detect cell survival rates of A549 cells treated with CHL1 siRNA and Akt inhibitor SC66. *P<0.05. (C) Colony formation assays were performed in A549 cells treated with CHL1 siRNA and the Akt inhibitor SC66 in the presence of DDP (1.5 µg/ml) or PTX (35 ng/ml). *P<0.05 vs. si-CHL1. (D) Apoptosis were measured in A549 cells treated with CHL1 siRNA and Akt inhibitor SC66 in the presence of DDP (1.5 µg/ml) and PTX (35 ng/ml). *P<0.05 vs. si-CHL1. CHL1, close homolog of L1; DDP, cisplatin; PTX, paclitaxel; si, small interfering; p-, phosphorylated.

Article Snippet: The resistant cells A549/PTX and A549/DDP cells were transfected with 4.0 µg CHL1 recombinant expression plasmid (cat. no. HG10143-NY; Sino Biological, Inc.).

Techniques: Activity Assay, Western Blot, Expressing, Plasmid Preparation

Journal: Molecular cell

Article Title: The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2

doi: 10.1016/j.molcel.2018.12.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human: CHL-1 , ATCC , CRL-9446.

Techniques: Western Blot, Transduction, In Situ, Virus, Recombinant, Lysis, Protease Inhibitor, Staining, Magnetic Beads, Cloning, Bicinchoninic Acid Protein Assay, Isolation, Labeling, Viability Assay, RNA Sequencing, Sequencing, Gene Expression, CRISPR, Mass Spectrometry, Expressing, Mutagenesis, shRNA, Plasmid Preparation, Control, Luciferase, Software, Membrane, Blocking Assay

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A ) Western blot analysis of CD63 EV marker in precipitated EV samples. The precipitated EVs derived using ExoQuick Solution were prepared from the serum collected from 2 mice of wild-type (WT) and EML4-ALK transgenic (TG) mouse as described in the “ Materials and methods ”. ( B ) Analysis of EMARS products obtained from EMARS reaction for crude mouse serum EVs. EMARS reaction was carried out directly in the precipitated serum EVs from two of WT and TG mice with or without HRP-conjugated CHL1 probe. The EMARS products were subsequently subjected to SDS-PAGE (10% gel) with fluorescein detection and CBB staining. ( C ) Fractionation using Sephacryl S-500 chromatography. Blue dextran (an indicator of void volume) and mouse serum (an indicator of protein elution) were used for preliminary experiments. The dotted line indicates absorbance of blue dextran. The solid line indicates protein concentration measured using a BCA protein kit. ( D ) Morphological observation of serum EVs (fraction No. 6 and No. 8) using cryo-electron microscopy. Lower panel of fraction No.6 is an enlarged view of a part of the upper panel. Scale bar; 200 nm (upper panel) and 50 nm (lower panel).

Article Snippet: In human CHL1 ELISA, the recombinant human CHL1 partial protein (10143-H08H; Sino Biological, Beijing, China) was used as a standard.

Techniques: Western Blot, Marker, Derivative Assay, Transgenic Assay, SDS Page, Staining, Fractionation, Chromatography, Protein Concentration, Electron Microscopy

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A ) Protein expression of precipitated EVs. The serum collected from seven mice of wild-type (WT) and EML4-ALK transgenic mouse (TL group) was subjected to Western blot analysis with anti-CHL1, anti-α2 integrin, anti-β1 integrin, and anti-FGFR3 antibodies, which were cancer cell membrane BiCAT molecules previously reported. ( B ) Western blot analysis for TSG101 antigen detection in Sephacryl S-500 fractions. The fractions were concentrated with Nanosep ® centrifugal unit, and then subjected to Western blot analysis with anti-TSG101 antibody. TSG101-(Ub)n indicates ubiquitinated TSG101.

Article Snippet: In human CHL1 ELISA, the recombinant human CHL1 partial protein (10143-H08H; Sino Biological, Beijing, China) was used as a standard.

Techniques: Expressing, Transgenic Assay, Western Blot

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A ) The EMARS products for serum EVs from EML4-ALK transgenic mouse. To average experimental results over each group, an aliquot of the serum (10 μL each) from 10 animals in each group (WT), large lung tumor-bearing (TL), and small lung tumor-bearing mice (TS) was mixed in equal proportions, and then applied to EV purification and EMARS. The EMARS products were concentrated and purified by immunoprecipitation with the anti-fluorescein antibody Sepharose. The resulting samples were subjected to SDS-PAGE analysis with fluorescence detection. “IP” indicates the immunoprecipitated samples, and “Lys” indicates the lysate samples before immunoprecipitation. The right panel indicates the same gel as the left panel, but exposed for a longer time. ( B ) Confirmation of candidate partner molecules (CD5L and PZP) with mouse CHL1 in EVs. The EMARS products of WT, TL, and TS were respectively applied to immunoprecipitation (anti-fluorescence antibody Sepharose) and western blot analysis with anti-CD5L (left panel) antibody. After the stripping as described in the “ Materials and methods ”, the membranes were re-stained with anti-PZP antibody (right panel). Arrows indicate the detected band of CD5L and PZP proteins (including predicted dimer). Asterisk indicates unknown bands (predicted as non-specific or partial fragments). ( C, D ) Confirmation of candidate partner molecules (SLC4A1 and THBS1) with mouse CHL1 in EVs. The western blot analysis was performed with anti-SLC4A1 antibody ( C ). After stripping, the membranes were re-stained with anti-THBS1 antibody ( D ). Arrows indicate the detected band of SLC4A1 and THBS1 proteins (including predicted dimers). Asterisks indicate unknown bands (predicted as non-specific or partial fragments). ( E ) Expression of SLC4A1 (left column) and CHL1 proteins (right column) in tumor tissues from two male and two female EML4-ALK transgenic mice. The fragments of lung cancer tissues were mashed and washed gently with PBS, and then lysed with SDS-PAGE sample buffer directly. The resulting samples were subjected to Western blot analysis with anti-SLC4A1 antibody and anti-CHL1 antibody. Arrows indicate the detected band of monomer SLC4A1 and CHL1 proteins.

Article Snippet: In human CHL1 ELISA, the recombinant human CHL1 partial protein (10143-H08H; Sino Biological, Beijing, China) was used as a standard.

Techniques: Transgenic Assay, Purification, Immunoprecipitation, SDS Page, Fluorescence, Western Blot, Stripping Membranes, Staining, Expressing

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A, B ) Western blot analysis of TSG101 ( A ) and CD63 ( B ) in serum EVs from WT, TL, and TS using anti-TSG101 and CD63 antibody. Arrows indicate the detected band of monomer TSG101 and CD63. The black bar indicates wide range of molecular weight due to an ubiquitination in TSG101 (TSG101-(Ub)n). ( C ) Western blot analysis of EMARS products with anti-CHL1 antibody. The EMARS products were concentrated and purified by immunoprecipitation with the anti-fluorescein antibody Sepharose. The resulting samples were subjected to SDS-PAGE analysis with fluorescence detection. “IP” indicates the immunoprecipitated samples, and “Lys” indicates the lysate samples before immunoprecipitation. Arrow indicates the detected band of CHL1. Asterisk indicates unknown bands (predicted as non-specific or partial fragments).

Article Snippet: In human CHL1 ELISA, the recombinant human CHL1 partial protein (10143-H08H; Sino Biological, Beijing, China) was used as a standard.

Techniques: Western Blot, Molecular Weight, Purification, Immunoprecipitation, SDS Page, Fluorescence

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A, B ) Calibration curve of sandwich ELISA for the detection of both SLC4A1 partial proteins and fluorescein-labeled SLC4A1. The detection of several concentrations of recombinant SLC4A1 partial protein using HRP-labeled anti-SLC4A1 antibody which is prepared using Zenon system is summarized in ( A ). The detection of several concentrations of self-made standard materials containing fluorescein-labeled SLC4A1 using HRP-labeled anti-fluorescein antibody is summarized in ( B ). ( C ) Comparison of serum CHL1 levels between wild-type (WT) and small tumor-bearing EML4-ALK transgenic (TS) mice by using previously established ELISA system for CHL1 measurement. There were no significant differences between them.

Article Snippet: In human CHL1 ELISA, the recombinant human CHL1 partial protein (10143-H08H; Sino Biological, Beijing, China) was used as a standard.

Techniques: Sandwich ELISA, Labeling, Recombinant, Transgenic Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A ) Calibration curve of sandwich ELISA for the detection of both human CHL1. The detection of several concentrations of recombinant human CHL1 partial protein using HRP-labeled anti-CHL1 antibody. ( B ) Comparison of serum CHL1 levels between H (open bar) and LC (closed bar) by using ELISA system for human CHL1 measurement. There were no significant differences between them. ( C ) Comparison of CHL1-expressing EVs between H (open bar) and LC (closed bar). The serum EVs were purified by using ExoQuick Solution followed by ELISA measurement of CHL1 levels. There were also no significant differences between them.

Article Snippet: In human CHL1 ELISA, the recombinant human CHL1 partial protein (10143-H08H; Sino Biological, Beijing, China) was used as a standard.

Techniques: Sandwich ELISA, Recombinant, Labeling, Enzyme-linked Immunosorbent Assay, Expressing, Purification

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A ) EMARS products purified from serum EVs of healthy person (H) and lung cancer (LC) patients. Fifty microliters of mouse serums was collected from the H and LC groups, and utilized in EV purification followed by EMARS reactions. To average experimental results over each group, an aliquot of the serum (10 μL each) from 5 H and 5 LC was mixed each in equal proportions. The EMARS products were subjected to SDS-PAGE analysis with fluorescence detection. ( B ) Confirmation of caspase 14 as a partner molecule with CHL1 identified by MS proteomics. The H and LC samples were applied respectively to immunoprecipitation (anti-fluorescence antibody Sepharose) and western blot analysis with anti-caspase 14 antibodies. Arrows indicate the detected band of caspase 14 proteins (including predicted dimer). ( C ) Measurement of fluorescein-labeled caspase 14 using a sandwich ELISA. Serum EVs from 12 H (open bar) and 12 LC (closed bar) were applied to EMARS reactions followed by ELISA measurements, respectively. The EMARS products containing fluorescein-labeled caspase 14 were added to anti-caspase 14 antibody-coated ELISA plates. “BiEV index (caspase 14)” was calculated based on the value of fluorescein-labeled recombinant caspase 14 made by fluorescein-labeling regent. The values are shown as the average of three independent ELISA experiments using the same samples. The detail data of H and LC persons is provided in Table S3. Asterisks indicate the samples were below detection limit. ( D ) ROC curve for BiEV indexes. The AUC was calculated as 0.811. ( E ) Western blot analysis of caspase 14 in whole-serum EVs from H and LC. An aliquot of the serum (2 μL each) from 12 persons in H and LC was mixed in equal proportions followed by EV purification with precipitation protocol. Arrows indicate the detected band of caspase 14.

Article Snippet: In human CHL1 ELISA, the recombinant human CHL1 partial protein (10143-H08H; Sino Biological, Beijing, China) was used as a standard.

Techniques: Purification, SDS Page, Fluorescence, Immunoprecipitation, Western Blot, Labeling, Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A ) Western blot analysis of CD63 EV marker in precipitated EV samples. The precipitated EVs derived using ExoQuick Solution were prepared from the serum collected from 2 mice of wild-type (WT) and EML4-ALK transgenic (TG) mouse as described in the “ Materials and methods ”. ( B ) Analysis of EMARS products obtained from EMARS reaction for crude mouse serum EVs. EMARS reaction was carried out directly in the precipitated serum EVs from two of WT and TG mice with or without HRP-conjugated CHL1 probe. The EMARS products were subsequently subjected to SDS-PAGE (10% gel) with fluorescein detection and CBB staining. ( C ) Fractionation using Sephacryl S-500 chromatography. Blue dextran (an indicator of void volume) and mouse serum (an indicator of protein elution) were used for preliminary experiments. The dotted line indicates absorbance of blue dextran. The solid line indicates protein concentration measured using a BCA protein kit. ( D ) Morphological observation of serum EVs (fraction No. 6 and No. 8) using cryo-electron microscopy. Lower panel of fraction No.6 is an enlarged view of a part of the upper panel. Scale bar; 200 nm (upper panel) and 50 nm (lower panel).

Article Snippet: The mouse and human anti-CHL1 antibodies (AF2147 and MAB2126; R&D systems, MN) were partially reduced and bound to HRP using a peroxidase labeling kit SH (Dojindo, Kumamoto, Japan).

Techniques: Western Blot, Marker, Derivative Assay, Transgenic Assay, SDS Page, Staining, Fractionation, Chromatography, Protein Concentration, Cryo-Electron Microscopy

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A ) Protein expression of precipitated EVs. The serum collected from seven mice of wild-type (WT) and EML4-ALK transgenic mouse (TL group) was subjected to Western blot analysis with anti-CHL1, anti-α2 integrin, anti-β1 integrin, and anti-FGFR3 antibodies, which were cancer cell membrane BiCAT molecules previously reported. ( B ) Western blot analysis for TSG101 antigen detection in Sephacryl S-500 fractions. The fractions were concentrated with Nanosep ® centrifugal unit, and then subjected to Western blot analysis with anti-TSG101 antibody. TSG101-(Ub)n indicates ubiquitinated TSG101.

Article Snippet: The mouse and human anti-CHL1 antibodies (AF2147 and MAB2126; R&D systems, MN) were partially reduced and bound to HRP using a peroxidase labeling kit SH (Dojindo, Kumamoto, Japan).

Techniques: Expressing, Transgenic Assay, Western Blot, Membrane

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A ) The EMARS products for serum EVs from EML4-ALK transgenic mouse. To average experimental results over each group, an aliquot of the serum (10 μL each) from 10 animals in each group (WT), large lung tumor-bearing (TL), and small lung tumor-bearing mice (TS) was mixed in equal proportions, and then applied to EV purification and EMARS. The EMARS products were concentrated and purified by immunoprecipitation with the anti-fluorescein antibody Sepharose. The resulting samples were subjected to SDS-PAGE analysis with fluorescence detection. “IP” indicates the immunoprecipitated samples, and “Lys” indicates the lysate samples before immunoprecipitation. The right panel indicates the same gel as the left panel, but exposed for a longer time. ( B ) Confirmation of candidate partner molecules (CD5L and PZP) with mouse CHL1 in EVs. The EMARS products of WT, TL, and TS were respectively applied to immunoprecipitation (anti-fluorescence antibody Sepharose) and western blot analysis with anti-CD5L (left panel) antibody. After the stripping as described in the “ Materials and methods ”, the membranes were re-stained with anti-PZP antibody (right panel). Arrows indicate the detected band of CD5L and PZP proteins (including predicted dimer). Asterisk indicates unknown bands (predicted as non-specific or partial fragments). ( C, D ) Confirmation of candidate partner molecules (SLC4A1 and THBS1) with mouse CHL1 in EVs. The western blot analysis was performed with anti-SLC4A1 antibody ( C ). After stripping, the membranes were re-stained with anti-THBS1 antibody ( D ). Arrows indicate the detected band of SLC4A1 and THBS1 proteins (including predicted dimers). Asterisks indicate unknown bands (predicted as non-specific or partial fragments). ( E ) Expression of SLC4A1 (left column) and CHL1 proteins (right column) in tumor tissues from two male and two female EML4-ALK transgenic mice. The fragments of lung cancer tissues were mashed and washed gently with PBS, and then lysed with SDS-PAGE sample buffer directly. The resulting samples were subjected to Western blot analysis with anti-SLC4A1 antibody and anti-CHL1 antibody. Arrows indicate the detected band of monomer SLC4A1 and CHL1 proteins.

Article Snippet: The mouse and human anti-CHL1 antibodies (AF2147 and MAB2126; R&D systems, MN) were partially reduced and bound to HRP using a peroxidase labeling kit SH (Dojindo, Kumamoto, Japan).

Techniques: Transgenic Assay, Purification, Immunoprecipitation, SDS Page, Fluorescence, Western Blot, Stripping Membranes, Staining, Expressing

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A, B ) Western blot analysis of TSG101 ( A ) and CD63 ( B ) in serum EVs from WT, TL, and TS using anti-TSG101 and CD63 antibody. Arrows indicate the detected band of monomer TSG101 and CD63. The black bar indicates wide range of molecular weight due to an ubiquitination in TSG101 (TSG101-(Ub)n). ( C ) Western blot analysis of EMARS products with anti-CHL1 antibody. The EMARS products were concentrated and purified by immunoprecipitation with the anti-fluorescein antibody Sepharose. The resulting samples were subjected to SDS-PAGE analysis with fluorescence detection. “IP” indicates the immunoprecipitated samples, and “Lys” indicates the lysate samples before immunoprecipitation. Arrow indicates the detected band of CHL1. Asterisk indicates unknown bands (predicted as non-specific or partial fragments).

Article Snippet: The mouse and human anti-CHL1 antibodies (AF2147 and MAB2126; R&D systems, MN) were partially reduced and bound to HRP using a peroxidase labeling kit SH (Dojindo, Kumamoto, Japan).

Techniques: Western Blot, Molecular Weight, Ubiquitin Proteomics, Purification, Immunoprecipitation, SDS Page, Fluorescence

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A, B ) Calibration curve of sandwich ELISA for the detection of both SLC4A1 partial proteins and fluorescein-labeled SLC4A1. The detection of several concentrations of recombinant SLC4A1 partial protein using HRP-labeled anti-SLC4A1 antibody which is prepared using Zenon system is summarized in ( A ). The detection of several concentrations of self-made standard materials containing fluorescein-labeled SLC4A1 using HRP-labeled anti-fluorescein antibody is summarized in ( B ). ( C ) Comparison of serum CHL1 levels between wild-type (WT) and small tumor-bearing EML4-ALK transgenic (TS) mice by using previously established ELISA system for CHL1 measurement. There were no significant differences between them.

Article Snippet: The mouse and human anti-CHL1 antibodies (AF2147 and MAB2126; R&D systems, MN) were partially reduced and bound to HRP using a peroxidase labeling kit SH (Dojindo, Kumamoto, Japan).

Techniques: Sandwich ELISA, Labeling, Recombinant, Comparison, Transgenic Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A ) Calibration curve of sandwich ELISA for the detection of both human CHL1. The detection of several concentrations of recombinant human CHL1 partial protein using HRP-labeled anti-CHL1 antibody. ( B ) Comparison of serum CHL1 levels between H (open bar) and LC (closed bar) by using ELISA system for human CHL1 measurement. There were no significant differences between them. ( C ) Comparison of CHL1-expressing EVs between H (open bar) and LC (closed bar). The serum EVs were purified by using ExoQuick Solution followed by ELISA measurement of CHL1 levels. There were also no significant differences between them.

Article Snippet: The mouse and human anti-CHL1 antibodies (AF2147 and MAB2126; R&D systems, MN) were partially reduced and bound to HRP using a peroxidase labeling kit SH (Dojindo, Kumamoto, Japan).

Techniques: Sandwich ELISA, Recombinant, Labeling, Comparison, Enzyme-linked Immunosorbent Assay, Expressing, Purification

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A ) EMARS products purified from serum EVs of healthy person (H) and lung cancer (LC) patients. Fifty microliters of mouse serums was collected from the H and LC groups, and utilized in EV purification followed by EMARS reactions. To average experimental results over each group, an aliquot of the serum (10 μL each) from 5 H and 5 LC was mixed each in equal proportions. The EMARS products were subjected to SDS-PAGE analysis with fluorescence detection. ( B ) Confirmation of caspase 14 as a partner molecule with CHL1 identified by MS proteomics. The H and LC samples were applied respectively to immunoprecipitation (anti-fluorescence antibody Sepharose) and western blot analysis with anti-caspase 14 antibodies. Arrows indicate the detected band of caspase 14 proteins (including predicted dimer). ( C ) Measurement of fluorescein-labeled caspase 14 using a sandwich ELISA. Serum EVs from 12 H (open bar) and 12 LC (closed bar) were applied to EMARS reactions followed by ELISA measurements, respectively. The EMARS products containing fluorescein-labeled caspase 14 were added to anti-caspase 14 antibody-coated ELISA plates. “BiEV index (caspase 14)” was calculated based on the value of fluorescein-labeled recombinant caspase 14 made by fluorescein-labeling regent. The values are shown as the average of three independent ELISA experiments using the same samples. The detail data of H and LC persons is provided in Table S3. Asterisks indicate the samples were below detection limit. ( D ) ROC curve for BiEV indexes. The AUC was calculated as 0.811. ( E ) Western blot analysis of caspase 14 in whole-serum EVs from H and LC. An aliquot of the serum (2 μL each) from 12 persons in H and LC was mixed in equal proportions followed by EV purification with precipitation protocol. Arrows indicate the detected band of caspase 14.

Article Snippet: The mouse and human anti-CHL1 antibodies (AF2147 and MAB2126; R&D systems, MN) were partially reduced and bound to HRP using a peroxidase labeling kit SH (Dojindo, Kumamoto, Japan).

Techniques: Purification, SDS Page, Fluorescence, Immunoprecipitation, Western Blot, Labeling, Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: bioRxiv

Article Title: Close homologue of L1 sensitizes lung cancer cells to cisplatin and paclitaxel via inhibition Akt pathway

doi: 10.1101/747238

Figure Lengend Snippet: (A) Cell survival of A549 and A549 resistant cells (A549/DDP, A549/PTX) treated with increasing concentration of DDP and PTX were measured by MTT assay. (B) The IC 50 values of DDP in A549/DDP, A549 cells, and the IC 50 values of PTX in A549/PTX, A549 cells. (C) Western blot assays dectected the expression of drug resistance-related genes MDR1, MRP and LRP in A549 cells and A549 resistant cells (A549/DDP, A549/PTX). The expression of CHL1 expression in A549 cells and A549 resistant cells (A549/DDP, A549/PTX) were analysed by Western blot (D) and qRT-PCR assays (E) respectively. (F) The expression of CHL1 mRNA in GSE21656 data. All results were expressed as mean ± SD, and were performed in triplicate. * P < 0.05.

Article Snippet: After blocked by 5% BSA for 2 h at 4°C, the membrane was incubated with primary antibodies anainst CHL1 (1:500, proteintech), MDR1 (1:500, proteintech), MRP (1:500, proteintech), LRP(1:500, proteintech), phospho-Akt(1:1000, Abcam), Akt (1:2000, Abcam), overnight at 4°C.

Techniques: Concentration Assay, MTT Assay, Western Blot, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Close homologue of L1 sensitizes lung cancer cells to cisplatin and paclitaxel via inhibition Akt pathway

doi: 10.1101/747238

Figure Lengend Snippet: (A) Western blot assay was performed to detect CHL1 interference efficiency in A549 cells transfected with CHL1 siRNAs. MTT assays were performed to determine survival rate of CHL1 knockdown A549 cells treated with 0–2.5 μg/ml DDP (B) or 0–50 ng/ml DDP (C). (D) Colony formation assay of A549 cells transfected with CHL1 siRNA in the presence or absence of 1.5 μg/ml DDP and 35 ng/ml PTX. (E) Flow cytometry analysis was used to detect apoptosis in A549 cells transfected with CHL1 siRNA in the presence or absence of 1.5 μg/ml DDP and 35 ng/ml PTX. All results were expressed as mean ± SD of three replications. * P < 0.05, *** P < 0.001.

Article Snippet: After blocked by 5% BSA for 2 h at 4°C, the membrane was incubated with primary antibodies anainst CHL1 (1:500, proteintech), MDR1 (1:500, proteintech), MRP (1:500, proteintech), LRP(1:500, proteintech), phospho-Akt(1:1000, Abcam), Akt (1:2000, Abcam), overnight at 4°C.

Techniques: Western Blot, Transfection, Colony Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Close homologue of L1 sensitizes lung cancer cells to cisplatin and paclitaxel via inhibition Akt pathway

doi: 10.1101/747238

Figure Lengend Snippet: (A) Western blot assay was performed to detect CHL1 expression in A549/DDP and A549/PTX cells transfected with CHL1 expression plasmids. Effect of CHL1 overexpression on A549 resistant cell survival rate when treated with 0-10 μg/ml DDP (B) or 0-250 ng/ml PTX (C). were determined by MTT assay. (D) Colony formation assays showed the number of colonies of A549 resistant cells transfected with CHL1 expression plasmids in the presence or absence of 8 μg/ml DDP and 160 ng/ml PTX. (E) Flow cytometry analysis was used to assess cell apoptosis in A549 resistant cells transfected with CHL1 expression plasmids in the presence or absence of 8 μg/ml DDP and 160 ng/ml PTX. All data represent the mean ± SD of three replications. * P < 0.05.

Article Snippet: After blocked by 5% BSA for 2 h at 4°C, the membrane was incubated with primary antibodies anainst CHL1 (1:500, proteintech), MDR1 (1:500, proteintech), MRP (1:500, proteintech), LRP(1:500, proteintech), phospho-Akt(1:1000, Abcam), Akt (1:2000, Abcam), overnight at 4°C.

Techniques: Western Blot, Expressing, Transfection, Over Expression, MTT Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Close homologue of L1 sensitizes lung cancer cells to cisplatin and paclitaxel via inhibition Akt pathway

doi: 10.1101/747238

Figure Lengend Snippet: (A) Western blot assay was performed to detect the expression of p-Akt and total Akt in CHL1 silenced and restored cell models. (B) MTT assays were performed to detect cell survival rates of A549 cells treated with CHL1 siRNA and Akt inhibitor SC66. (D) Colony formation assays were excuted in A549 cells treated with CHL1 siRNA and Akt inhibitor SC66 in presence DDP (1.5 μg/mL) and PTX (35 ng/ml). (E) Cell apoptosis were measured in A549 cells treated with CHL1 siRNA and Akt inhibitor SC66 in presence DDP (1.5 μg/mL) and PTX (35 ng/ml). All data represent the mean ± SD of three replications. * P < 0.05.

Article Snippet: After blocked by 5% BSA for 2 h at 4°C, the membrane was incubated with primary antibodies anainst CHL1 (1:500, proteintech), MDR1 (1:500, proteintech), MRP (1:500, proteintech), LRP(1:500, proteintech), phospho-Akt(1:1000, Abcam), Akt (1:2000, Abcam), overnight at 4°C.

Techniques: Western Blot, Expressing

Journal: Experimental and Therapeutic Medicine

Article Title: CHL1 inhibits cell proliferation, migration and invasion by regulating the NF‑κB signaling pathway in colorectal cancer

doi: 10.3892/etm.2024.12454

Figure Lengend Snippet: CHL1 inhibits colorectal cancer cell migration and invasion. (A and B) Transwell assay was performed to measure the (A) migratory and (B) invasive ability of HT29 and SW480 cells that were transfected with pcDNA3.1-CHL1 vector (CHL1) and NC. (C) Immunofluorescence labeling of E-cadherin protein in HT29 and SW480 cells. (D) Western blotting was used to detect E-cadherin and N-cadherin protein levels in HT29 and SW480 cells. ** P<0.01 and *** P<0.001 vs. the NC group. CHL1, cell adhesion molecule close homolog of L1; NC, normal control.

Article Snippet: After blocking the membrane with 5% skimmed milk for 1 h at 25˚C, it was incubated overnight with primary antibody E-cadherin (cat. no. 20874-1-AP, Proteintech Group, Inc., dilution 1:20,000), N-cadherin (cat. no. 22018-1-AP, Proteintech Group, Inc., dilution 1:2,000), CHL1 (cat. no. 25250-1-AP, Proteintech Group, Inc., dilution 1:500), p-p65 (cat. no. AP0475; ABclonal, Inc., dilution 1:500), p65 (cat. no. 10745-1-AP, Proteintech Group, Inc., dilution 1:2,000) and GAPDH (cat. no. 10494-1-AP, Proteintech Group, Inc., dilution 1:6,000) at 4˚C.

Techniques: Migration, Transwell Assay, Transfection, Plasmid Preparation, Immunofluorescence, Labeling, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: CHL1 inhibits cell proliferation, migration and invasion by regulating the NF‑κB signaling pathway in colorectal cancer

doi: 10.3892/etm.2024.12454

Figure Lengend Snippet: CHL1 inhibits colorectal cancer cell migration and invasion by regulating the TNF-α/NF-κB pathway. (A and B) Transwell assay was conducted to detect cell migration and invasion. (C) Immunofluorescence assay for positive protein expression of E-cadherin. CHL1 and RELA (p65) overexpression plasmid or NC plasmid were simultaneously transfected for 2 days and then used for this assay. ** P<0.01, and *** P<0.001 vs. the NC group. # P<0.05, ## P<0.01 and ### P<0.001 vs. the CHL1 group. CHL1, cell adhesion molecule close homolog of L1; NC, normal control.

Article Snippet: After blocking the membrane with 5% skimmed milk for 1 h at 25˚C, it was incubated overnight with primary antibody E-cadherin (cat. no. 20874-1-AP, Proteintech Group, Inc., dilution 1:20,000), N-cadherin (cat. no. 22018-1-AP, Proteintech Group, Inc., dilution 1:2,000), CHL1 (cat. no. 25250-1-AP, Proteintech Group, Inc., dilution 1:500), p-p65 (cat. no. AP0475; ABclonal, Inc., dilution 1:500), p65 (cat. no. 10745-1-AP, Proteintech Group, Inc., dilution 1:2,000) and GAPDH (cat. no. 10494-1-AP, Proteintech Group, Inc., dilution 1:6,000) at 4˚C.

Techniques: Migration, Transwell Assay, Immunofluorescence, Expressing, Over Expression, Plasmid Preparation, Transfection