Journal: Frontiers in Microbiology

Article Title: Identification of a Novel Serum Biomarker for Tuberculosis Infection in Chinese HIV Patients by iTRAQ-Based Quantitative Proteomics

doi: 10.3389/fmicb.2018.00330

Figure Lengend Snippet: Differentially expressed proteins in the sera of HIV positive patients with active tuberculosis (TB) relative to controls (only HIV positive patients).

Article Snippet: The concentrations of ENG (CUSABIO, CSB-E10030h, China), PSMB2 (CUSABIO, CSB-E17836h, China), HSP90AA1 (CUSABIO, CSB-E13462h, China), HSPA8 (CUSABIO, CSB-EL010829HU, China), CHI3L1 (CUSABIO, CSB-E13608h, China), CAP1 (CUSABIO, CSB-EL004486HU, China), ORM1 (CUSABIO, CSB-EL017237HU, China), RETN (CUSABIO, CSB-E06884h, China), LPA (CUSABIO, CSB-EQ028005HU, China) and ZYX (CUSABIO, CSB-EL027165HU, China) in the serum samples were quantified using ELISA according to the manufacturer’s instructions.

Techniques:

Journal: Theranostics

Article Title: Chitinase-3 like-protein-1 promotes glioma progression via the NF-κB signaling pathway and tumor microenvironment reprogramming

doi: 10.7150/thno.75069

Figure Lengend Snippet: CHI3L1 expression is correlated with the malignancy and OS of glioma . (A-B) The mRNA levels of CHI3L1 in 33 cancer types and paired normal tissues in TCGA. (C-E) CHI3L1 expression levels in normal and glioma tissues in GEO, TCGA and CGGA. (F-H) CHI3L1 expression levels in staged gliomas in GEO, TCGA and CGGA. (G) CGGA mRNA_array_301, (H) CGGA mRNAseq_325. (I) Serum levels of CHI3L1 in patients with glioma. (J) Representative pictures of H&E staining and IHC analysis of CHI3L1 expression levels in glioma and paired normal tissues; Scale bars represent 100 µm. (K-L) Western blot and quantitative analysis of CHI3L1 expression in seven pairs of glioma with different grades and paired normal tissues. (M-N) Western blot and quantitative analysis of CHI3L1 expression in normal tissues and staged gliomas. (O-P) ROC curve exhibited the sensitivity and specificity of CHI3L1 to predict glioma in TCGA and CGGA databases. (O) 5 y (AUC = 0.819), 3 y (AUC = 0.881), 1 y (AUC = 0.852). (P) 5 y (AUC = 0.784), 3 y (AUC = 0.782), 1 y (AUC = 0.741), y: year. TPR: true positive rate. FPR: False positive rate. (Q-S) Survival benefits between CHI3L1 high and CHI3L1 low groups (median was the cut-off to identify high and low groups) were assessed via Kaplan-Meier analysis using both the Log-rank and Wilcoxon-Breslow tests. (Q) Log-rank p = 0, HR (high) = 6.4, p (HR) = 0, n (high) = 338, n (low) = 337. (R) Log-rank p < 0.0001, Wilcoxon p < 0.0001. (S) Log-rank p = 0.0692, Wilcoxon p = 0.0243. (T) The mRNA expression profile of CHI3L1 in glioma cell lines from the CCLE. (U-V) Comparison of CHI3L1 expression levels in glioma cell lines analyzed by one-way analysis of variance (ANOVA). N: normal. G: glioma. T: tumors. OS: overall survival. PFS: progression-free survival. M: months. Student's t test, *p < 0.05; **p < 0.01; ****p < 0.0001.

Article Snippet: The recombinant human CHI3L1 (rhCHI3L1) was purchased from Sino Biological lnc (Human, Cat. #11227-H08H).

Techniques: Expressing, Staining, Western Blot

Journal: Theranostics

Article Title: Chitinase-3 like-protein-1 promotes glioma progression via the NF-κB signaling pathway and tumor microenvironment reprogramming

doi: 10.7150/thno.75069

Figure Lengend Snippet: CHI3L1 expression is associated with NF-κB pathway activation in glioma. (A-B) Volcano plot and heatmap of differentially expressed genes (DEGs) in GSE100675 dataset. (C-D) GO and GSEA based on DEGs identified in GSE100675. (E) Heatmap of genes targeting the NF-κB pathway in normal and tumors. (F-G) Volcano plot and heatmap of DEGs in TCGA glioma cohort. (H) Heatmap of 50 hallmark gene sets performed by GSVA in TCGA. (I-J) Enrichment score of TNFɑ NF-κB pathway among normal and tumor, as well as among CHI3L1 high and CHI3L1 low groups in TCGA, Wilcoxon test. (K) Spearman correlation analysis between the mRNA expression of CHI3L1 and NF-κB pathway enrichment score in TCGA. t student (163) = 13.48, r Pearson = 0.73, CI 95% [0.64, 0.79], n pairs = 165. log e (BF 01 ) = -57.89, = 0.72, = [0.64, 0.79], = 1.41. (L-M) CHI3L1 expression and TNFɑ NF-κB pathway enrichment score between the five subclasses of glioma in TCGA, Kruskal-wallis test. (N) Heatmap of 50 hallmark gene sets by GSVA in CGGA mRNA-array 301 dataset. (O) Density plot showed the TNFɑ NF-κB pathway enrichment score between CHI3L1 high and CHI3L1 low groups in CGGA, Wilcoxon test. (P) Spearman correlation analysis between CHI3L1 expression and NF-κB pathway enrichment score in CGGA. t student (299) = 16.69, r Pearson = 0.69, CI 95% [0.63, 0.75], n pairs = 301. log e (BF 01 ) = -95.06, = 0.69, = [0.63, 0.75], = 1.41. (Q-R) Violin plot showed the expression of CHI3L1 and TNFɑ NF-κB pathway enrichment score between the four subclasses of glioma in CGGA, Kruskal-wallis test. (Q) F welch (3, 90.33) = 101.56, p = 7.72e-29, CI 95% [0.69, 1.00], n obs = 301. (R) F welch (3, 89.73) = 103.07, p = 5.70e-29, CI 95% [0.70, 1.00], n obs = 301. RES: Ranked Enrichment Score. RLM: Ranked List Metric. RiOD: Rank in Ordered Dataset.

Article Snippet: The recombinant human CHI3L1 (rhCHI3L1) was purchased from Sino Biological lnc (Human, Cat. #11227-H08H).

Techniques: Expressing, Activation Assay

Journal: Theranostics

Article Title: Chitinase-3 like-protein-1 promotes glioma progression via the NF-κB signaling pathway and tumor microenvironment reprogramming

doi: 10.7150/thno.75069

Figure Lengend Snippet: CHI3L1 high specific glioma cells dominantly drive the activation of the NF-κB pathway . UMAP plot of total cells from patients with staged gliomas, with each cell color coded for cluster (A) , cell type (C) , and sample origin (E-F) . (B) Violin plot showed marker genes for cell annotation. (D) CHI3L1 expression profiles in all cells in the UMAP plot. (G) UMAP plot of all cells in tumoral and non-tumoral regions coded for cell type. (H) Heatmap of 50 hallmark gene sets in eight identified cell clusters using singscore. (I-K) Unsupervised transcriptional trajectory of CHI3L1 high and CHI3L1 low glioma cells, coded by cell type, state, and pseudotime. (L) UMAP dot plot of CHI3L1 expression levels in gliomas. (M-N) Pseudotime trajectory of CHI3L1 high and CHI3L1 low glioma cells in the UMAP dot plot. (O) Dynamic changes in CHI3L1 expression levels in all glioma cell types and across pseudotime. (P) The correlation between expression of CHI3L1 and NF-κB pathway enrichment score. t student (1954) = 21.22, r Pearson = 0.43, CI 95% [0.40, 0.47], n pairs = 1956. log e (BF 01 ) = -198.95, = 0.43, = [0.40, 0.47], = 1.41. Bc: brain cell, Mφ: Macrophage, Mic: Microglia, Mon: Monocyte, Unk: Unknown, Neu: Neutrophil, Tc: Tumor cell. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: The recombinant human CHI3L1 (rhCHI3L1) was purchased from Sino Biological lnc (Human, Cat. #11227-H08H).

Techniques: Activation Assay, Marker, Expressing

Journal: Theranostics

Article Title: Chitinase-3 like-protein-1 promotes glioma progression via the NF-κB signaling pathway and tumor microenvironment reprogramming

doi: 10.7150/thno.75069

Figure Lengend Snippet: CHI3L1 binds to ACTN4 and NFKB1, and promote the activation of NF-κB pathway . (A) Schematic of the procedure used to detect biotin-hCHI3L1-binding proteins using HuProt 20K human proteome microarrays. (B) ACTN1, 4, NFKB1, 2, and NFKBIB were identified as CHI3L1-binding proteins in the proteome microarrays. (C) The IF analysis of the expression levels of CHI3L1 and NF-κB p65 subunit in peritumor and intratumor regions; Scale bars represent 100 µm. (D) Western blot analysis of the expression levels of CHI3L1 in U118MG and U251MG cells after treated with TNFɑ (200 ng/mL) for 0-96 h. (E) Chi3l1 +/+ and Chi3l1 -/- BMDMs were treated with TNFɑ (50 and 200 ng/mL), and phosphorylation of p65 were assessed via western blot. (F-G) U87MG and A172 cells transfected with shCtrl and shCHI3L1 were treated with TNFɑ, and the phosphorylation of p65 were detected using western blot. (H-I) Cytoplasmic and nuclear proteins were isolated from BMDMs and U87MG after treated with TNFɑ (50 ng/mL) and applied to western blot to detect the expression of p65 and ACTN4. (J) Lysates from U87MG and A172 cells were immunoprecipitated with IgG or anti-CHI3L1 antibody, and then immunoblotted as indicated. (K) Cytoplasmic and nuclear proteins were isolated from U87MG cells. The lysates were immunoprecipitated with IgG or anti-CHI3L1 antibody, and then immunoblotted as indicated. (L-N) Co-localization of CHI3L1 and ACTN4, NFKB1, or p65 in U87MG or A172 cells observed by confocal microscope. (O) CHI3L1 and ACTN4 enhance NF-kB activation by using a dual-luciferase reporter assay. (P) Dox-inducible CHI3L1 expression enhanced enhanced NF-kB activation by TNFα in U251 cells. (Q) Pearson correlations between CHI3L1 expression and ACTN4 , NFKB1 , NFKB2 , RelA (p65), and NFKBIB in TCGA and CGGA glioma cohorts.

Article Snippet: The recombinant human CHI3L1 (rhCHI3L1) was purchased from Sino Biological lnc (Human, Cat. #11227-H08H).

Techniques: Activation Assay, Binding Assay, Expressing, Western Blot, Transfection, Isolation, Immunoprecipitation, Microscopy, Luciferase, Reporter Assay

Journal: Theranostics

Article Title: Chitinase-3 like-protein-1 promotes glioma progression via the NF-κB signaling pathway and tumor microenvironment reprogramming

doi: 10.7150/thno.75069

Figure Lengend Snippet: CHI3L1 reprogrammed the TME to an immunosuppressive phenotype in glioma . (A-B) Heatmap and violin plot showed the immune cells infiltration in glioma in TCGA. (C-D) Total macrophages and lymphocytes comparison between normal and glioma tissues in TCGA, Wilcoxon test. (E) Spearman correlation analysis between the CHI3L1 expression and immune cells infiltration in TCGA. (F) The immune score, estimate score, stromal score, tumor purity, TIS score, dysfunction, exclusion and TIDE score between the CHI3L1 high and CHI3L1 low groups of patients with glioma in TCGA, Wilcoxon test. (G-J) Spearman correlation analysis between the expression of CHI3L1 and immune checkpoints in glioma, as well as in MES glioma in TCGA. (H) t student (163) = 7.09, r Pearson = 0.49, CI 95% [0.36, 0.59], n pairs = 165. log e (BF 01 ) = -19.44, = 0.48, = [0.37, 0.60], = 1.41. (K-N) Spearman correlation analysis between the expression of CHI3L1 and immune checkpoints in glioma, as well as in MES glioma in CGGA. (L) t student (299) = 8.89, r Pearson = 0.46, CI 95% [0.36, 0.54], n pairs = 301. log e (BF 01 ) = -32.33, = 0.45, = [0.37, 0.54], = 1.41. (O-P) Flow cytometry analysis of the mean fluorescence intensity (MFI) of CD274 (PDL1) staining in U251 (O) and U118 (P) cells transfected with empty vector or CHI3L1 expression plasmid. act.: activated, Neu: neutrophils, Mφ: macrophages, Mon: monocytes, DC: dendritic cells, Tregs: regulatory T cells, Eos: eosinophils, rest.: resting, Tfh: T cells follicular helper.

Article Snippet: The recombinant human CHI3L1 (rhCHI3L1) was purchased from Sino Biological lnc (Human, Cat. #11227-H08H).

Techniques: Expressing, Flow Cytometry, Fluorescence, Staining, Transfection, Plasmid Preparation

Journal: Theranostics

Article Title: Chitinase-3 like-protein-1 promotes glioma progression via the NF-κB signaling pathway and tumor microenvironment reprogramming

doi: 10.7150/thno.75069

Figure Lengend Snippet: Myeloid landscape delineation in human glioma. UMAP plot of monocyte, microglia and macrophage populations (A) , with each cell color coded for cluster (B) . (C) Heatmap of hallmark gene sets with significant difference using singscore. (D-F) Pseudotime trajectory of monocyte, microglia, and macrophage state transition inferred by Monocle 2 and characterized by cell type (D) , state (E) , and pseudotime (F) . (G-H) Trajectory of monocytes, microglia, and macrophage state transition in UMPA plot. (I-L) Dynamic changes of the CHI3L1 and CD44 expression during the state transition profile coded for cell type (I) , cluster (K) , and pseudotime (J, L) . (M) UMAP dot plot of neutrophil from 13 patients, with each cell color coded for cluster. (N) Expression profile of CHI3L1 in neutrophils in the UMAP plot. (O) Heatmap of hallmark gene sets with significant difference in neutrophils performed by singscore. (P-R) Trajectory analysis of neutrophils annotated by cluster, state and pseudotime. (S-T) Expression profiles (S) and dynamic changes (T) of CHI3L1 among subclusters of neutrophils. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: The recombinant human CHI3L1 (rhCHI3L1) was purchased from Sino Biological lnc (Human, Cat. #11227-H08H).

Techniques: Expressing

Journal: Theranostics

Article Title: Chitinase-3 like-protein-1 promotes glioma progression via the NF-κB signaling pathway and tumor microenvironment reprogramming

doi: 10.7150/thno.75069

Figure Lengend Snippet: CHI3L1 interacts with CD44 to drive M2 TAMs polarization. (A-B) Western blot of CHI3L1, p65, and ACTN4 expression after TNFɑ treatment in U87MG and A172 cells, which were pretreated with dimethyl sulfoxide (DMSO), CB, (-)-B, PF, and Y. (C-D) Cytoplasmic and nuclear CHI3L1, p65, and ACTN4 were analyzed using western blot. (E) mRNA expression levels of CHI3L1 between the control and CB pretreated group in U87MG and A172 cell lines. (F) Co-immunofluorescent staining of CHI3L1 and CD206 in frozen sections of human gliomas. (G-H) Macrophages derived from THP1 induced by PMA (10 ng/mL) for 24 h, and identified by morphologic evaluation and mRNA expression of CD68. (I) M2 markers (CD206 and CD163) expression in macrophages treated with IL-4 (100 ng/mL), rhCHI3L1 (500 ng/mL), and the culture supernatant (cs) of U87MG and A172 cells measured by IF. (J-K) mRNA expression of M1 and M2 markers quantitated by qRT-PCR in macrophages treated with IL4, and the culture supernatant of U87MG and A172 cells. (L-N) Migration of U118MG cells induced by M2 macrophages. (O-V) mRNA expression of M1 and M2 markers in Cd44 +/+ and Cd44 -/- BMDMs treated with IL-4 and rmCHI3L1 (500 ng/mL). (W) Western blot of phosphorylation of AKT and p38 in Cd44 +/+ and Cd44 -/- BMDMs pretreated with rmCHI3L1.

Article Snippet: The recombinant human CHI3L1 (rhCHI3L1) was purchased from Sino Biological lnc (Human, Cat. #11227-H08H).

Techniques: Western Blot, Expressing, Staining, Derivative Assay, Quantitative RT-PCR, Migration

Journal: Theranostics

Article Title: Chitinase-3 like-protein-1 promotes glioma progression via the NF-κB signaling pathway and tumor microenvironment reprogramming

doi: 10.7150/thno.75069

Figure Lengend Snippet: CHI3L1 regulates the proliferation, migration and survival of glioma cells. (A-B) The mRNA expression of inflammatory factors in U87MG and A172 cells. (C-D) Colony formation assay was performed in U87MG cells. (E-F) Proliferation curve of CFSE labelled A172 cells. (G-I) Proliferation of U87MG and A172 cells evaluated by EdU incorporation assay. (J-M) Migration of U87MG and A172 cells evaluated by wound healing assay. (N) The infection efficacy of the lentivirus. (O) The knockdown efficiency of the shCHI3L1 lentrivirus. (P) Proliferations of U87MG cells with or without CHI3L1 knockdown evaluated by CCK-8 assay. Student's t test, *p < 0.05, compared with the shCHI3L1 group. (Q) The growth rates and size of the control tumors and the tumors with CHI3L1 knockdown. Student's t test, *p < 0.05, compared with the shCHI3L1 group. (R) The working model of CHI3L1 on glioma cells and TME. Student's t test, *p < 0.05, *p < 0.01, compared with the shCtrl group.

Article Snippet: The recombinant human CHI3L1 (rhCHI3L1) was purchased from Sino Biological lnc (Human, Cat. #11227-H08H).

Techniques: Migration, Expressing, Colony Assay, Wound Healing Assay, Infection, CCK-8 Assay