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$FIGURE 1 BACE1 substrate identification in vivo. A, Proteomic workflow of the Stable Isotope Labeling by Amino acids (SILA) spike-in approach. Isotopically heavy (13C6) labeled mouse brain tissue was combined with unlabeled (light) mouse brain tissue from wild-type (WT) or BACE1 KO mice. WT and BACE1 KO mice were littermates. B, Volcano plot of membrane fractions of BACE1 KO and WT mouse brains. The negative log10 transformed P value (y-axis) is plotted against the mean protein log2 fold change (x-axis) between BACE1 KO and WT samples. Single-span transmembrane proteins and GPI-anchored proteins are colored in blue. The hyperbolic curves represent a permutation-based FDR correction for multiple hypotheses. Proteins above the FDR curve remain significantly changed after FDR correction. C, Western blot analysis of the membrane preparation of WT, and BACE1 KO mouse brains at P3 shows accumulation of full-length CNTN2, <t>CHL1,</t> APP, MDGA1, and CACHD1. D, Statistical analysis was performed with n = 8 biological replicates using Mann-Whitney U test with the significance criteria of P < .05. All proteins accumulate significantly, except for CACHD1 that shows a nonsignificant trend to an increase. Graphs are presented with mean ± SEM$
Chl1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 63 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rat+anti+human+chl1+antibody/Human%2FMouse%2FRat+Contactin-2%2FTAG1+Antibody/pm31908000-73-20-21
Average 93 stars, based on 63 article reviews

chl1 - by Bioz Stars, 2026-09

93/100 stars

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1) Product Images from "Mouse brain proteomics establishes MDGA1 and CACHD1 as in vivo substrates of the Alzheimer protease BACE1."

Article Title: Mouse brain proteomics establishes MDGA1 and CACHD1 as in vivo substrates of the Alzheimer protease BACE1.

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

doi: 10.1096/fj.201902347R

$FIGURE 1 BACE1 substrate identification in vivo. A, Proteomic workflow of the Stable Isotope Labeling by Amino acids (SILA) spike-in approach. Isotopically heavy (13C6) labeled mouse brain tissue was combined with unlabeled (light) mouse brain tissue from wild-type (WT) or BACE1 KO mice. WT and BACE1 KO mice were littermates. B, Volcano plot of membrane fractions of BACE1 KO and WT mouse brains. The negative log10 transformed P value (y-axis) is plotted against the mean protein log2 fold change (x-axis) between BACE1 KO and WT samples. Single-span transmembrane proteins and GPI-anchored proteins are colored in blue. The hyperbolic curves represent a permutation-based FDR correction for multiple hypotheses. Proteins above the FDR curve remain significantly changed after FDR correction. C, Western blot analysis of the membrane preparation of WT, and BACE1 KO mouse brains at P3 shows accumulation of full-length CNTN2, CHL1, APP, MDGA1, and CACHD1. D, Statistical analysis was performed with n = 8 biological replicates using Mann-Whitney U test with the significance criteria of P < .05. All proteins accumulate significantly, except for CACHD1 that shows a nonsignificant trend to an increase. Graphs are presented with mean ± SEM$
Figure Legend Snippet: FIGURE 1 BACE1 substrate identification in vivo. A, Proteomic workflow of the Stable Isotope Labeling by Amino acids (SILA) spike-in approach. Isotopically heavy (13C6) labeled mouse brain tissue was combined with unlabeled (light) mouse brain tissue from wild-type (WT) or BACE1 KO mice. WT and BACE1 KO mice were littermates. B, Volcano plot of membrane fractions of BACE1 KO and WT mouse brains. The negative log10 transformed P value (y-axis) is plotted against the mean protein log2 fold change (x-axis) between BACE1 KO and WT samples. Single-span transmembrane proteins and GPI-anchored proteins are colored in blue. The hyperbolic curves represent a permutation-based FDR correction for multiple hypotheses. Proteins above the FDR curve remain significantly changed after FDR correction. C, Western blot analysis of the membrane preparation of WT, and BACE1 KO mouse brains at P3 shows accumulation of full-length CNTN2, CHL1, APP, MDGA1, and CACHD1. D, Statistical analysis was performed with n = 8 biological replicates using Mann-Whitney U test with the significance criteria of P < .05. All proteins accumulate significantly, except for CACHD1 that shows a nonsignificant trend to an increase. Graphs are presented with mean ± SEM

Techniques Used: In Vivo, Quantitative Proteomics, Labeling, Membrane, Transformation Assay, Western Blot, MANN-WHITNEY

Incubation:

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells
Article Snippet: .. Then, sections were blocked with 10% normal goat serum in PBS at room temperature for 30 min, and samples were subjected to incubation with the following primary antibodies: rat anti-human CHL1 antibody (1:100, cat. no. MAB2126, R&D Systems), rabbit polyclonal anti-PCNA antibody (1:200, cat. no. sc-7907, Santa Cruz), rabbit polyclonal anti-caspase-3 antibody (1:200, cat. no. sc-7148, Santa Cruz), rabbit polyclonal anti-GFAP antibody (1:500, cat. no. BA0056, Boster Biological Technology) at 4°C overnight. ..

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells.
Article Snippet: .. Then, sections were blocked with 10% normal goat serum in PBS at room temperature for 30 min, and samples were subjected to incubation with the following primary antibodies: rat anti-human CHL1 antibody (1:100, cat. no. MAB2126, R&D Systems), rabbit polyclonal anti-PCNA antibody (1:200, cat. no. sc-7907, Santa Cruz), rabbit polyclonal anti-caspase-3 antibody (1:200, cat. no. sc-7148, Santa Cruz), rabbit polyclonal anti-GFAP antibody (1:500, cat. no. BA0056, Boster Biological Technology) at 4◦C overnight. ..

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells.
Article Snippet: .. Membranes were incubated with a rat anti-human CHL1 antibody that specifically targets the extracellular domain of CHL1 (1:500, R&D Systems, cat. no. MAB2126, Minneapolis, MN, USA), mouse monoclonal anti-Bcl-2 antibody (1:1000, cat. no. sc-7382, Santa Cruz, CA, USA), rabbit polyclonal anti-Bax antibody (1:1000, cat. no. sc-526, Santa Cruz), rabbit polyclonal anti-PCNA antibody (1:1000, cat. no. sc-7907, Santa Cruz), rabbit polyclonal anti-caspase-3 antibody (1:1000, cat. no. sc-7148, Santa Cruz), mouse monoclonal anti phosphorylated extracellular signal regulated kinase 1/2 (anti-pErk1/2) antibody (1:1000, cat. no. sc-7383, Santa Cruz), mouse monoclonal antiErk1/2 antibody (1:1000, cat. no. sc-135900, Santa Cruz), mouse monoclonal anti-pAkt1 antibody (1:1000, cat. no. sc-81433, Santa Cruz), mouse monoclonal anti-Akt1 antibody (1:1000, cat. no. sc-55523, Santa Cruz) and mouse monoclonal antiglyceraldehyde-3-phosphate dehydrogenase (GADPH) antibody (1:1000, cat. no. sc-365062, Santa Cruz) overnight at 4◦C. ..

Image Search Results

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Mouse brain proteomics establishes MDGA1 and CACHD1 as in vivo substrates of the Alzheimer protease BACE1.

doi: 10.1096/fj.201902347R

Figure Lengend Snippet: FIGURE 1 BACE1 substrate identification in vivo. A, Proteomic workflow of the Stable Isotope Labeling by Amino acids (SILA) spike-in approach. Isotopically heavy (13C6) labeled mouse brain tissue was combined with unlabeled (light) mouse brain tissue from wild-type (WT) or BACE1 KO mice. WT and BACE1 KO mice were littermates. B, Volcano plot of membrane fractions of BACE1 KO and WT mouse brains. The negative log10 transformed P value (y-axis) is plotted against the mean protein log2 fold change (x-axis) between BACE1 KO and WT samples. Single-span transmembrane proteins and GPI-anchored proteins are colored in blue. The hyperbolic curves represent a permutation-based FDR correction for multiple hypotheses. Proteins above the FDR curve remain significantly changed after FDR correction. C, Western blot analysis of the membrane preparation of WT, and BACE1 KO mouse brains at P3 shows accumulation of full-length CNTN2, CHL1, APP, MDGA1, and CACHD1. D, Statistical analysis was performed with n = 8 biological replicates using Mann-Whitney U test with the significance criteria of P < .05. All proteins accumulate significantly, except for CACHD1 that shows a nonsignificant trend to an increase. Graphs are presented with mean ± SEM

Article Snippet: Primary antibodies used: APP (Millipore, Merck; 22C11, dilution 1:1000), BACE1 (Robert Vassar, Northwestern University, Chicago, IL, USA; 3D5, dilution 1:1000), CHL1 (R&D Systems, Minneapolis, MN, USA; AF2147, dilution | 5NJAVRO et Al. 1:1000), CNTN2 (R&D Systems; AF4439, dilution 1:1000), β-actin (Sigma, Merck; AC-74, A5316, dilution 1:1000), Calnexin (Enzo Life Sciences, Germany; ADI-SPA-860, dilution 1:1000), HA (Covance, Princeton, NJ, USA; MMS101P, dilution 1:1000), NrCAM (Abcam, Cambridge, UK; ab24344, dilution 1:1000), and SEZ6 (dilution 1:10 of primary hybridoma supernatant).47 Monoclonal anti-CACHD1 antibody CACHI 15H10 (rat IgG1/k) (dilution 1:10 of primary hybridoma supernatant) was generated by immunization of Lou/c rats with the peptide NLENDRDERDDDSHEDR (intracellular part of murine and human CACHD1) using standard procedures.48 Monoclonal anti-MDGA1 antibody DGA 21E3 (rat IgG2a/k) (dilution 1:10 of primary hybridoma supernatant) was generated by immunization of Lou/c rats with native recombinant, C-terminally BAP-HIS-tagged murine MDGA1 ectodomain spanning amino acids 20-925 which was lentivirally transduced and overexpressed in HEK293T via a Gal4-UAS expression system and purified via metal affinity chromatography.

Techniques: In Vivo, Quantitative Proteomics, Labeling, Membrane, Transformation Assay, Western Blot, MANN-WHITNEY

Sequences for random control siRNA and siRNAs against CHL1.

Journal: Frontiers in Molecular Neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: Sequences for random control siRNA and siRNAs against CHL1.

Article Snippet: Then, sections were blocked with 10% normal goat serum in PBS at room temperature for 30 min, and samples were subjected to incubation with the following primary antibodies: rat anti-human CHL1 antibody (1:100, cat. no. MAB2126, R&D Systems), rabbit polyclonal anti-PCNA antibody (1:200, cat. no. sc-7907, Santa Cruz), rabbit polyclonal anti-caspase-3 antibody (1:200, cat. no. sc-7148, Santa Cruz), rabbit polyclonal anti-GFAP antibody (1:500, cat. no. BA0056, Boster Biological Technology) at 4°C overnight.

Techniques: Control, Sequencing

Western blot analysis of the protein levels of CHL1 detected in normal human glial HEB cells and 3 glioma/glioblastoma cell lines. CHL1 was weakly expressed in normal human HEB glial cells. Its levels in all the 3 glioma/glioblastoma cells were higher than that in normal human HEB glial cells, with the statistical significance detected in SHG44 cells (* p < 0.05 vs. HEB cells) and U-87 MG cells (** p < 0.01 vs. HEB cells). n = 3 for each group. Student’s t -test for independent samples was used.

Journal: Frontiers in Molecular Neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: Western blot analysis of the protein levels of CHL1 detected in normal human glial HEB cells and 3 glioma/glioblastoma cell lines. CHL1 was weakly expressed in normal human HEB glial cells. Its levels in all the 3 glioma/glioblastoma cells were higher than that in normal human HEB glial cells, with the statistical significance detected in SHG44 cells (* p < 0.05 vs. HEB cells) and U-87 MG cells (** p < 0.01 vs. HEB cells). n = 3 for each group. Student’s t -test for independent samples was used.

Techniques: Western Blot

Treatment of siRNA targeting CHL1 in three human glioma cell lines. Total RNA was isolated from U251, SHG44 and U-87 MG cells treated with vehicle control (vc), control siRNA (control siRNA) or siRNA targeting CHL1 (CHL1 siRNA). RT-PCR and Western blot analysis were then used to measure both relative mRNA and protein levels of CHL1. (A) RT-PCR analysis of the mRNA levels of CHL1 in U251, SHG44 and U-87 MG cells treated with vehicle control (vc), control siRNA and siRNA targeting CHL1, and (B) Western blot analysis of the protein levels of CHL1 detected in U251, SHG44 and U-87 MG cells treated with vehicle control (vc), control siRNA and siRNA targeting CHL1. Data are presented as means ± standard error of the mean (SEM) ( n = 3, * p < 0.05; ** p < 0.01, independent Student’s t -test).

Journal: Frontiers in Molecular Neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: Treatment of siRNA targeting CHL1 in three human glioma cell lines. Total RNA was isolated from U251, SHG44 and U-87 MG cells treated with vehicle control (vc), control siRNA (control siRNA) or siRNA targeting CHL1 (CHL1 siRNA). RT-PCR and Western blot analysis were then used to measure both relative mRNA and protein levels of CHL1. (A) RT-PCR analysis of the mRNA levels of CHL1 in U251, SHG44 and U-87 MG cells treated with vehicle control (vc), control siRNA and siRNA targeting CHL1, and (B) Western blot analysis of the protein levels of CHL1 detected in U251, SHG44 and U-87 MG cells treated with vehicle control (vc), control siRNA and siRNA targeting CHL1. Data are presented as means ± standard error of the mean (SEM) ( n = 3, * p < 0.05; ** p < 0.01, independent Student’s t -test).

Techniques: Isolation, Control, Reverse Transcription Polymerase Chain Reaction, Western Blot

Knockdown of CHL1 affects the proliferation and survival of U251, SHG44 and U-87 MG glioma cells. Cells were seeded on 96-well plates in triplicate, and proliferation rates were measured by MTT assay to evaluate the effect of CHL1 on the proliferation of U251, SHG44 and U-87 MG glioma/glioblastoma cells. (A–C) Changes of the proliferation rate in U251 (A) , SHG44 (B) , and U-87 MG (C) cells treated with vehicle control (vc), control siRNA (control siRNA) or siRNA targeting CHL1 (CHL1 siRNA). The data were expressed as the means ± SEM of three independent experiments (* p < 0.05 and ** p < 0.01 vs. either vehicle control or control siRNA; independent Student’s t -test).

Journal: Frontiers in Molecular Neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: Knockdown of CHL1 affects the proliferation and survival of U251, SHG44 and U-87 MG glioma cells. Cells were seeded on 96-well plates in triplicate, and proliferation rates were measured by MTT assay to evaluate the effect of CHL1 on the proliferation of U251, SHG44 and U-87 MG glioma/glioblastoma cells. (A–C) Changes of the proliferation rate in U251 (A) , SHG44 (B) , and U-87 MG (C) cells treated with vehicle control (vc), control siRNA (control siRNA) or siRNA targeting CHL1 (CHL1 siRNA). The data were expressed as the means ± SEM of three independent experiments (* p < 0.05 and ** p < 0.01 vs. either vehicle control or control siRNA; independent Student’s t -test).

Techniques: Knockdown, MTT Assay, Control

Knockdown of CHL1 affects the senescence of glioma/glioblastoma cells in vitro . (A–C) U251 (A) , SHG44 (B) and U-87 MG (C) cells were seeded onto 24-well plates and treated with vehicle control, control siRNA and siRNA targeting CHL1, and senescent cells were then detected by senescence-associated β-galactosidase staining (200×). The data were expressed as the means ± SEM from four independent experiments (** p < 0.01; *** p < 0.001 vs. either vehicle control or control siRNA; Independent Student’s t -test).

Journal: Frontiers in Molecular Neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: Knockdown of CHL1 affects the senescence of glioma/glioblastoma cells in vitro . (A–C) U251 (A) , SHG44 (B) and U-87 MG (C) cells were seeded onto 24-well plates and treated with vehicle control, control siRNA and siRNA targeting CHL1, and senescent cells were then detected by senescence-associated β-galactosidase staining (200×). The data were expressed as the means ± SEM from four independent experiments (** p < 0.01; *** p < 0.001 vs. either vehicle control or control siRNA; Independent Student’s t -test).

Techniques: Knockdown, In Vitro, Control, Staining

Knockdown of CHL1 reduced colony formation capacity of glioma/glioblastoma cells in vitro . Cell colony was stained by crystal violet, which was then dissolved in 1% SDS and the optical density was measured at 546 nm under a microplate reader. (A) The colony formation assay revealed that knockdown of CHL1 reduced the colony formation of U251 cells, as was revealed by the optical density detected at 546 nm from three independent experiments (* p < 0.05; ** p < 0.01 vs. either vehicle control or control siRNA). (B,C) Similar results were found in SHG44 (B) and U-87 MG (C) cells for the colony formation experiment. The data were expressed as the means ± SEM from 4 independent experiments (* p < 0.05 and ** p < 0.01 vs. both vehicle control and control siRNA; independent Student’s t -test).

Journal: Frontiers in Molecular Neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: Knockdown of CHL1 reduced colony formation capacity of glioma/glioblastoma cells in vitro . Cell colony was stained by crystal violet, which was then dissolved in 1% SDS and the optical density was measured at 546 nm under a microplate reader. (A) The colony formation assay revealed that knockdown of CHL1 reduced the colony formation of U251 cells, as was revealed by the optical density detected at 546 nm from three independent experiments (* p < 0.05; ** p < 0.01 vs. either vehicle control or control siRNA). (B,C) Similar results were found in SHG44 (B) and U-87 MG (C) cells for the colony formation experiment. The data were expressed as the means ± SEM from 4 independent experiments (* p < 0.05 and ** p < 0.01 vs. both vehicle control and control siRNA; independent Student’s t -test).

Techniques: Knockdown, In Vitro, Staining, Colony Assay, Control

Knockdown of CHL1 suppressed the migration of glioma/glioblastoma cells in vitro . Transwell migration assays were carried out using U251 (A) , SHG44 (B) and U-87 MG (C) cells transfected with CHL siRNA. Representative fields containing migrated cells attached to the underside of the membrane were presented. The migration ability was indexed by the relative number of migrated cells from three independent experiments. The data were expressed as the means ± SEM from 3 independent experiments (* p < 0.05; ** p < 0.01 vs. both vehicle control and control siRNA (independent Student’s t -test).

Journal: Frontiers in Molecular Neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: Knockdown of CHL1 suppressed the migration of glioma/glioblastoma cells in vitro . Transwell migration assays were carried out using U251 (A) , SHG44 (B) and U-87 MG (C) cells transfected with CHL siRNA. Representative fields containing migrated cells attached to the underside of the membrane were presented. The migration ability was indexed by the relative number of migrated cells from three independent experiments. The data were expressed as the means ± SEM from 3 independent experiments (* p < 0.05; ** p < 0.01 vs. both vehicle control and control siRNA (independent Student’s t -test).

Techniques: Knockdown, Migration, In Vitro, Transfection, Membrane, Control

Knockdown of CHL1 affects apoptosis signaling molecules in glioma/glioblastoma cells. U251, SHG44 and U-87 MG cells were seeded onto 48-well plates and treated with vehicle control, control siRNA and siRNA targeting CHL1, respectively. Western blot analysis was performed to determine the levels the apoptosis-related proteins, including changes of the ratio of Bax to Bcl-2 (Bax/Bcl-2) (A) , active caspase-3 (B) and PCNA (C) in glioma/glioblastoma cells. GAPDH was used as the loading control. The data were expressed as the means ± SEM from three independent experiments (* p < 0.05; ** p < 0.01; *** p < 0.001 vs. either vehicle control or control siRNA; independent Student’s t -test).

Journal: Frontiers in Molecular Neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: Knockdown of CHL1 affects apoptosis signaling molecules in glioma/glioblastoma cells. U251, SHG44 and U-87 MG cells were seeded onto 48-well plates and treated with vehicle control, control siRNA and siRNA targeting CHL1, respectively. Western blot analysis was performed to determine the levels the apoptosis-related proteins, including changes of the ratio of Bax to Bcl-2 (Bax/Bcl-2) (A) , active caspase-3 (B) and PCNA (C) in glioma/glioblastoma cells. GAPDH was used as the loading control. The data were expressed as the means ± SEM from three independent experiments (* p < 0.05; ** p < 0.01; *** p < 0.001 vs. either vehicle control or control siRNA; independent Student’s t -test).

Techniques: Knockdown, Control, Western Blot

Knockdown of CHL1 reduced the phosphorylation levels of ERK and AKT. Western blot was used to analyze the levels of pAkt and pErk in three cell lines after treatment with vehicle control, negative control and CHL1 siRNA for 48 h. pAkt and pErk protein levels in U251 (A) , SHG44 (B) and U-87 MG cells (C) were presented. GAPDH was used as a loading control. The data were expressed as the means ± SEM from 3 independent experiments (* p < 0.05 and ** p < 0.01 vs. both vehicle control and control siRNA; independent Students t -test).

Journal: Frontiers in Molecular Neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: Knockdown of CHL1 reduced the phosphorylation levels of ERK and AKT. Western blot was used to analyze the levels of pAkt and pErk in three cell lines after treatment with vehicle control, negative control and CHL1 siRNA for 48 h. pAkt and pErk protein levels in U251 (A) , SHG44 (B) and U-87 MG cells (C) were presented. GAPDH was used as a loading control. The data were expressed as the means ± SEM from 3 independent experiments (* p < 0.05 and ** p < 0.01 vs. both vehicle control and control siRNA; independent Students t -test).

Techniques: Knockdown, Phospho-proteomics, Western Blot, Control, Negative Control

CHL1 regulates growth of U-87 MG glioma cells in vivo . (A,B) Two weeks after the 1st intratumoral injection, all mice were killed by cervical dislocation. The in situ tumors and the dissected tumor tissues were photographed. (C) The fold increase of volume at each day points post the 1st intratumoral injection of either control siRNA or CHL1 siRNA complexed with the Entranster™- in vivo . (D) Column diagram showing the final average tumor volumes from both control siRNA and CHL1 siRNA-treated group ( n = 5, p = 0.2768 vs. the control siRNA group) (* p < 0.05; ** p < 0.01 vs. control siRNA; Independent Student’s t -test).

Journal: Frontiers in Molecular Neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: CHL1 regulates growth of U-87 MG glioma cells in vivo . (A,B) Two weeks after the 1st intratumoral injection, all mice were killed by cervical dislocation. The in situ tumors and the dissected tumor tissues were photographed. (C) The fold increase of volume at each day points post the 1st intratumoral injection of either control siRNA or CHL1 siRNA complexed with the Entranster™- in vivo . (D) Column diagram showing the final average tumor volumes from both control siRNA and CHL1 siRNA-treated group ( n = 5, p = 0.2768 vs. the control siRNA group) (* p < 0.05; ** p < 0.01 vs. control siRNA; Independent Student’s t -test).

Techniques: In Vivo, Injection, In Situ, Control

H&E staining and immunohistochemical staining analyses for the CHL1, caspase-3, PCNA and GFAP molecules in glioblastoma xenograft tissues from both control siRNA and CHL1 siRNA-treated groups. Scale bars represent 25 μm.

Journal: Frontiers in Molecular Neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: H&E staining and immunohistochemical staining analyses for the CHL1, caspase-3, PCNA and GFAP molecules in glioblastoma xenograft tissues from both control siRNA and CHL1 siRNA-treated groups. Scale bars represent 25 μm.

Techniques: Staining, Immunohistochemical staining, Control

FIGURE 1 | Western blot analysis of the protein levels of CHL1 detected in normal human glial HEB cells and 3 glioma/glioblastoma cell lines. CHL1 was weakly expressed in normal human HEB glial cells. Its levels in all the 3 glioma/glioblastoma cells were higher than that in normal human HEB glial cells, with the statistical signiﬁcance detected in SHG44 cells (∗p < 0.05 vs. HEB cells) and U-87 MG cells (∗∗p < 0.01 vs. HEB cells). n = 3 for each group. Student’s t-test for independent samples was used.

Journal: Frontiers in molecular neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells.

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: FIGURE 1 | Western blot analysis of the protein levels of CHL1 detected in normal human glial HEB cells and 3 glioma/glioblastoma cell lines. CHL1 was weakly expressed in normal human HEB glial cells. Its levels in all the 3 glioma/glioblastoma cells were higher than that in normal human HEB glial cells, with the statistical signiﬁcance detected in SHG44 cells (∗p < 0.05 vs. HEB cells) and U-87 MG cells (∗∗p < 0.01 vs. HEB cells). n = 3 for each group. Student’s t-test for independent samples was used.

Article Snippet: Membranes were incubated with a rat anti-human CHL1 antibody that specifically targets the extracellular domain of CHL1 (1:500, R&D Systems, cat. no. MAB2126, Minneapolis, MN, USA), mouse monoclonal anti-Bcl-2 antibody (1:1000, cat. no. sc-7382, Santa Cruz, CA, USA), rabbit polyclonal anti-Bax antibody (1:1000, cat. no. sc-526, Santa Cruz), rabbit polyclonal anti-PCNA antibody (1:1000, cat. no. sc-7907, Santa Cruz), rabbit polyclonal anti-caspase-3 antibody (1:1000, cat. no. sc-7148, Santa Cruz), mouse monoclonal anti phosphorylated extracellular signal regulated kinase 1/2 (anti-pErk1/2) antibody (1:1000, cat. no. sc-7383, Santa Cruz), mouse monoclonal antiErk1/2 antibody (1:1000, cat. no. sc-135900, Santa Cruz), mouse monoclonal anti-pAkt1 antibody (1:1000, cat. no. sc-81433, Santa Cruz), mouse monoclonal anti-Akt1 antibody (1:1000, cat. no. sc-55523, Santa Cruz) and mouse monoclonal antiglyceraldehyde-3-phosphate dehydrogenase (GADPH) antibody (1:1000, cat. no. sc-365062, Santa Cruz) overnight at 4◦C.

Techniques: Western Blot

FIGURE 2 | Treatment of siRNA targeting CHL1 in three human glioma cell lines. Total RNA was isolated from U251, SHG44 and U-87 MG cells treated with vehicle control (vc), control siRNA (control siRNA) or siRNA targeting CHL1 (CHL1 siRNA). RT-PCR and Western blot analysis were then used to measure both relative mRNA and protein levels of CHL1. (A) RT-PCR analysis of the mRNA levels of CHL1 in U251, SHG44 and U-87 MG cells treated with vehicle control (vc), control siRNA and siRNA targeting CHL1, and (B) Western blot analysis of the protein levels of CHL1 detected in U251, SHG44 and U-87 MG cells treated with vehicle control (vc), control siRNA and siRNA targeting CHL1. Data are presented as means ± standard error of the mean (SEM) (n = 3, ∗p < 0.05; ∗∗p < 0.01, independent Student’s t-test).

Journal: Frontiers in molecular neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells.

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: FIGURE 2 | Treatment of siRNA targeting CHL1 in three human glioma cell lines. Total RNA was isolated from U251, SHG44 and U-87 MG cells treated with vehicle control (vc), control siRNA (control siRNA) or siRNA targeting CHL1 (CHL1 siRNA). RT-PCR and Western blot analysis were then used to measure both relative mRNA and protein levels of CHL1. (A) RT-PCR analysis of the mRNA levels of CHL1 in U251, SHG44 and U-87 MG cells treated with vehicle control (vc), control siRNA and siRNA targeting CHL1, and (B) Western blot analysis of the protein levels of CHL1 detected in U251, SHG44 and U-87 MG cells treated with vehicle control (vc), control siRNA and siRNA targeting CHL1. Data are presented as means ± standard error of the mean (SEM) (n = 3, ∗p < 0.05; ∗∗p < 0.01, independent Student’s t-test).

Techniques: Isolation, Control, Reverse Transcription Polymerase Chain Reaction, Western Blot

FIGURE 3 | Knockdown of CHL1 affects the proliferation and survival of U251, SHG44 and U-87 MG glioma cells. Cells were seeded on 96-well plates in triplicate, and proliferation rates were measured by MTT assay to evaluate the effect of CHL1 on the proliferation of U251, SHG44 and U-87 MG glioma/glioblastoma cells. (A–C) Changes of the proliferation rate in U251 (A), SHG44 (B), and U-87 MG (C) cells treated with vehicle control (vc), control siRNA (control siRNA) or siRNA targeting CHL1 (CHL1 siRNA). The data were expressed as the means ± SEM of three independent experiments (∗p < 0.05 and ∗∗p < 0.01 vs. either vehicle control or control siRNA; independent Student’s t-test).

Journal: Frontiers in molecular neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells.

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: FIGURE 3 | Knockdown of CHL1 affects the proliferation and survival of U251, SHG44 and U-87 MG glioma cells. Cells were seeded on 96-well plates in triplicate, and proliferation rates were measured by MTT assay to evaluate the effect of CHL1 on the proliferation of U251, SHG44 and U-87 MG glioma/glioblastoma cells. (A–C) Changes of the proliferation rate in U251 (A), SHG44 (B), and U-87 MG (C) cells treated with vehicle control (vc), control siRNA (control siRNA) or siRNA targeting CHL1 (CHL1 siRNA). The data were expressed as the means ± SEM of three independent experiments (∗p < 0.05 and ∗∗p < 0.01 vs. either vehicle control or control siRNA; independent Student’s t-test).

Techniques: Knockdown, MTT Assay, Control

FIGURE 4 | Knockdown of CHL1 affects the senescence of glioma/glioblastoma cells in vitro. (A–C) U251 (A), SHG44 (B) and U-87 MG (C) cells were seeded onto 24-well plates and treated with vehicle control, control siRNA and siRNA targeting CHL1, and senescent cells were then detected by senescence-associated β-galactosidase staining (200×). The data were expressed as the means ± SEM from four independent experiments (∗∗p < 0.01; ∗∗∗p < 0.001 vs. either vehicle control or control siRNA; Independent Student’s t-test).

Journal: Frontiers in molecular neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells.

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: FIGURE 4 | Knockdown of CHL1 affects the senescence of glioma/glioblastoma cells in vitro. (A–C) U251 (A), SHG44 (B) and U-87 MG (C) cells were seeded onto 24-well plates and treated with vehicle control, control siRNA and siRNA targeting CHL1, and senescent cells were then detected by senescence-associated β-galactosidase staining (200×). The data were expressed as the means ± SEM from four independent experiments (∗∗p < 0.01; ∗∗∗p < 0.001 vs. either vehicle control or control siRNA; Independent Student’s t-test).

Techniques: Knockdown, In Vitro, Control, Staining

FIGURE 5 | Knockdown of CHL1 reduced colony formation capacity of glioma/glioblastoma cells in vitro. Cell colony was stained by crystal violet, which was then dissolved in 1% SDS and the optical density was measured at 546 nm under a microplate reader. (A) The colony formation assay revealed that knockdown of CHL1 reduced the colony formation of U251 cells, as was revealed by the optical density detected at 546 nm from three independent experiments (∗p < 0.05; ∗∗p < 0.01 vs. either vehicle control or control siRNA). (B,C) Similar results were found in SHG44 (B) and U-87 MG (C) cells for the colony formation experiment. The data were expressed as the means ± SEM from 4 independent experiments (∗p < 0.05 and ∗∗p < 0.01 vs. both vehicle control and control siRNA; independent Student’s t-test).

Journal: Frontiers in molecular neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells.

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: FIGURE 5 | Knockdown of CHL1 reduced colony formation capacity of glioma/glioblastoma cells in vitro. Cell colony was stained by crystal violet, which was then dissolved in 1% SDS and the optical density was measured at 546 nm under a microplate reader. (A) The colony formation assay revealed that knockdown of CHL1 reduced the colony formation of U251 cells, as was revealed by the optical density detected at 546 nm from three independent experiments (∗p < 0.05; ∗∗p < 0.01 vs. either vehicle control or control siRNA). (B,C) Similar results were found in SHG44 (B) and U-87 MG (C) cells for the colony formation experiment. The data were expressed as the means ± SEM from 4 independent experiments (∗p < 0.05 and ∗∗p < 0.01 vs. both vehicle control and control siRNA; independent Student’s t-test).

Techniques: Knockdown, In Vitro, Staining, Colony Assay, Control

FIGURE 6 | Knockdown of CHL1 suppressed the migration of glioma/glioblastoma cells in vitro. Transwell migration assays were carried out using U251 (A), SHG44 (B) and U-87 MG (C) cells transfected with CHL siRNA. Representative ﬁelds containing migrated cells attached to the underside of the membrane were presented. The migration ability was indexed by the relative number of migrated cells from three independent experiments. The data were expressed as the means ± SEM from 3 independent experiments (∗p < 0.05; ∗∗p < 0.01 vs. both vehicle control and control siRNA (independent Student’s t-test).

Journal: Frontiers in molecular neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells.

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: FIGURE 6 | Knockdown of CHL1 suppressed the migration of glioma/glioblastoma cells in vitro. Transwell migration assays were carried out using U251 (A), SHG44 (B) and U-87 MG (C) cells transfected with CHL siRNA. Representative ﬁelds containing migrated cells attached to the underside of the membrane were presented. The migration ability was indexed by the relative number of migrated cells from three independent experiments. The data were expressed as the means ± SEM from 3 independent experiments (∗p < 0.05; ∗∗p < 0.01 vs. both vehicle control and control siRNA (independent Student’s t-test).

Techniques: Knockdown, Migration, In Vitro, Transfection, Membrane, Control

FIGURE 7 | Knockdown of CHL1 affects apoptosis signaling molecules in glioma/glioblastoma cells. U251, SHG44 and U-87 MG cells were seeded onto 48-well plates and treated with vehicle control, control siRNA and siRNA targeting CHL1, respectively. Western blot analysis was performed to determine the levels the apoptosis-related proteins, including changes of the ratio of Bax to Bcl-2 (Bax/Bcl-2) (A), active caspase-3 (B) and PCNA (C) in glioma/glioblastoma cells. GAPDH was used as the loading control. The data were expressed as the means ± SEM from three independent experiments (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 vs. either vehicle control or control siRNA; independent Student’s t-test).

Journal: Frontiers in molecular neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells.

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: FIGURE 7 | Knockdown of CHL1 affects apoptosis signaling molecules in glioma/glioblastoma cells. U251, SHG44 and U-87 MG cells were seeded onto 48-well plates and treated with vehicle control, control siRNA and siRNA targeting CHL1, respectively. Western blot analysis was performed to determine the levels the apoptosis-related proteins, including changes of the ratio of Bax to Bcl-2 (Bax/Bcl-2) (A), active caspase-3 (B) and PCNA (C) in glioma/glioblastoma cells. GAPDH was used as the loading control. The data were expressed as the means ± SEM from three independent experiments (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 vs. either vehicle control or control siRNA; independent Student’s t-test).

Techniques: Knockdown, Control, Western Blot

FIGURE 8 | Knockdown of CHL1 reduced the phosphorylation levels of ERK and AKT. Western blot was used to analyze the levels of pAkt and pErk in three cell lines after treatment with vehicle control, negative control and CHL1 siRNA for 48 h. pAkt and pErk protein levels in U251 (A), SHG44 (B) and U-87 MG cells (C) were presented. GAPDH was used as a loading control. The data were expressed as the means ± SEM from 3 independent experiments (∗p < 0.05 and ∗∗p < 0.01 vs. both vehicle control and control siRNA; independent Students t-test).

Journal: Frontiers in molecular neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells.

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: FIGURE 8 | Knockdown of CHL1 reduced the phosphorylation levels of ERK and AKT. Western blot was used to analyze the levels of pAkt and pErk in three cell lines after treatment with vehicle control, negative control and CHL1 siRNA for 48 h. pAkt and pErk protein levels in U251 (A), SHG44 (B) and U-87 MG cells (C) were presented. GAPDH was used as a loading control. The data were expressed as the means ± SEM from 3 independent experiments (∗p < 0.05 and ∗∗p < 0.01 vs. both vehicle control and control siRNA; independent Students t-test).

Techniques: Knockdown, Phospho-proteomics, Western Blot, Control, Negative Control

FIGURE 9 | CHL1 regulates growth of U-87 MG glioma cells in vivo. (A,B) Two weeks after the 1st intratumoral injection, all mice were killed by cervical dislocation. The in situ tumors and the dissected tumor tissues were photographed. (C) The fold increase of volume at each day points post the 1st intratumoral injection of either control siRNA or CHL1 siRNA complexed with the EntransterTM-in vivo. (D) Column diagram showing the ﬁnal average tumor volumes from both control siRNA and CHL1 siRNA-treated group (n = 5, p = 0.2768 vs. the control siRNA group) (∗p < 0.05; ∗∗p < 0.01 vs. control siRNA; Independent Student’s t-test).

Journal: Frontiers in molecular neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells.

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: FIGURE 9 | CHL1 regulates growth of U-87 MG glioma cells in vivo. (A,B) Two weeks after the 1st intratumoral injection, all mice were killed by cervical dislocation. The in situ tumors and the dissected tumor tissues were photographed. (C) The fold increase of volume at each day points post the 1st intratumoral injection of either control siRNA or CHL1 siRNA complexed with the EntransterTM-in vivo. (D) Column diagram showing the ﬁnal average tumor volumes from both control siRNA and CHL1 siRNA-treated group (n = 5, p = 0.2768 vs. the control siRNA group) (∗p < 0.05; ∗∗p < 0.01 vs. control siRNA; Independent Student’s t-test).

Techniques: In Vivo, Injection, In Situ, Control

FIGURE 10 | H&E staining and immunohistochemical staining analyses for the CHL1, caspase-3, PCNA and GFAP molecules in glioblastoma xenograft tissues from both control siRNA and CHL1 siRNA-treated groups. Scale bars represent 25 µm.

Journal: Frontiers in molecular neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells.

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: FIGURE 10 | H&E staining and immunohistochemical staining analyses for the CHL1, caspase-3, PCNA and GFAP molecules in glioblastoma xenograft tissues from both control siRNA and CHL1 siRNA-treated groups. Scale bars represent 25 µm.

Techniques: Staining, Immunohistochemical staining, Control

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1) Product Images from "Mouse brain proteomics establishes MDGA1 and CACHD1 as in vivo substrates of the Alzheimer protease BACE1."

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