chi3l1  (R&D Systems)

Journal: PLoS ONE

Article Title: Stat3 Downstream Gene Product Chitinase 3-Like 1 Is a Potential Biomarker of Inflammation-induced Lung Cancer in Multiple Mouse Lung Tumor Models and Humans

doi: 10.1371/journal.pone.0061984

Figure Lengend Snippet: A) Expression of Stat3C-Flag in the whole lung of CCSP-rtTA/(tetO)7-CMV-Stat3C double-transgenic mice. Actin was used as control. WT: wild type mouse lungs; -Dox: doxycycline untreated bouble transgenic mouse lungs; +Dox1 & +Dox2: doxycycline treated mouse lungs. B) Expression of CHI3L1 protein in BALF of CCSP-rtTA/(TetO) 7 -CMV-Stat3C double-transgenic mice. Lane 1–3, doxycycline-untreated mice (Dox-); Lane 4–6, doxycycline-treated mice without showing lung tumor (Dox+); Lane 7–10, doxycycline-treated mice showing lung tumor (Lung Cancer).

Article Snippet: For CHI3L1 protein detection, BALFs from doxycycline-treated or untreated CCSP-rtTA/(TetO) 7 -CMV-Stat3 bitransgenic mice were collected as described above and diluted with Laemmli sample buffer (Bio-Rad, Hercules, CA) at 1∶1, then heated in boiling water for 5 min. Gel electrophoresis and antibody hybridization were performed following the same procedure as above except that rat anti-mouse ChI3L1 primary antibody (1∶1,000, R&D, Minneapolis, MN) and goat anti rat IgG secondary antibody (1∶3000, Vector Laboratories, Burlingame, CA) were used.

Techniques: Expressing, Transgenic Assay

Journal: PLoS ONE

Article Title: Stat3 Downstream Gene Product Chitinase 3-Like 1 Is a Potential Biomarker of Inflammation-induced Lung Cancer in Multiple Mouse Lung Tumor Models and Humans

doi: 10.1371/journal.pone.0061984

Figure Lengend Snippet: Left: CHI3L1 protein concentrations in BALF and serum of doxycycline-treated and untreated bitransgenic mice. Right: ROC (Receiver Operating Characteristic) curve analyses to determine the area under the curve (AUC). Dox-: doxycycline-untreated mice; Dox+: doxycycline-treated mice without showing lung tumor; Cancer, doxycycline-treated mice with lung tumor. Mean ± SD in BALF (n>13), DOX -: 8.8±10.0, DOX +: 100.0±49.0, Cancer: 151.8±67.3. Mean ± SD in serum (n>13), DOX -: 6.2±3.7, DOX +: 33.5±35.6, Cancer: 159.0±90.0. The gray lines represent the mean values.

Article Snippet: For CHI3L1 protein detection, BALFs from doxycycline-treated or untreated CCSP-rtTA/(TetO) 7 -CMV-Stat3 bitransgenic mice were collected as described above and diluted with Laemmli sample buffer (Bio-Rad, Hercules, CA) at 1∶1, then heated in boiling water for 5 min. Gel electrophoresis and antibody hybridization were performed following the same procedure as above except that rat anti-mouse ChI3L1 primary antibody (1∶1,000, R&D, Minneapolis, MN) and goat anti rat IgG secondary antibody (1∶3000, Vector Laboratories, Burlingame, CA) were used.

Techniques:

Journal: PLoS ONE

Article Title: Stat3 Downstream Gene Product Chitinase 3-Like 1 Is a Potential Biomarker of Inflammation-induced Lung Cancer in Multiple Mouse Lung Tumor Models and Humans

doi: 10.1371/journal.pone.0061984

Figure Lengend Snippet: Left: CHI3L1 protein concentrations in BALF and serum of doxycycline-treated and untreated bitransgenic mice. Right: ROC (Receiver Operating Characteristic) curve analyses to determine the area under the curve (AUC). Dox-: doxycycline-untreated mice; Dox+: doxycycline-treated mice without showing lung tumor; Cancer, doxycycline-treated mice with lung tumor. A) CCSP-rtTA/(TetO) 7 -CMV-MMP12 mice. Mean ± SD in BALF (n>7), DOX -: 49.6±22.8, DOX +: 49.4±18.1, Cancer: 159.7±22.4. Mean ± SD in serum (n>7), DOX -: 18.6±10.9, DOX +: 18.2±4.6, Cancer: 61.4±33.5. The gray lines represent the mean values; B) CCSP-rtTA/(TetO) 7 -CMV-Api6 mice. Mean ± SD in BALF (n>3), DOX -: 82.4±37.1, DOX +: 93.8±23.0, Cancer: 409.8±49.2. Mean ± SD in serum (n>3), DOX -: 14.0±7.2, DOX +: 30.7±43.1, Cancer: 295.2±194.0. The gray lines represent the mean values.

Article Snippet: For CHI3L1 protein detection, BALFs from doxycycline-treated or untreated CCSP-rtTA/(TetO) 7 -CMV-Stat3 bitransgenic mice were collected as described above and diluted with Laemmli sample buffer (Bio-Rad, Hercules, CA) at 1∶1, then heated in boiling water for 5 min. Gel electrophoresis and antibody hybridization were performed following the same procedure as above except that rat anti-mouse ChI3L1 primary antibody (1∶1,000, R&D, Minneapolis, MN) and goat anti rat IgG secondary antibody (1∶3000, Vector Laboratories, Burlingame, CA) were used.

Techniques:

Journal: PLoS ONE

Article Title: Stat3 Downstream Gene Product Chitinase 3-Like 1 Is a Potential Biomarker of Inflammation-induced Lung Cancer in Multiple Mouse Lung Tumor Models and Humans

doi: 10.1371/journal.pone.0061984

Figure Lengend Snippet: Left: CHI3L1 protein concentrations in BALF and serum of doxycycline-treated and untreated bitransgenic mice. Right: ROC (Receiver Operating Characteristic) curve analyses to determine the area under the curve (AUC). Dox-: doxycycline-untreated mice; Dox+: doxycycline-treated mice without showing lung tumor; Cancer, doxycycline-treated mice with lung tumor. A) c-fms-rtTA/(TetO) 7 -CMV-MMP12 mice. Mean ± SD in BALF (n>7), DOX -: 17.7±13.8, DOX +: 42.1±67.9, Cancer: 70.9±36.5. Mean ± SD in serum (n>7), DOX -: 39.1±25.6, DOX +: 57.7±55.5, Cancer: 232.3±110.6. The gray lines represent the mean values; B) c-fms-rtTA/(TetO) 7 -CMV-Api6 mice. Mean ± SD in BALF (n>6), DOX -: 69.6±16.6, DOX +: 73.2±28.1, Cancer: 189.9±47.2. Mean ± SD in serum (n>6), DOX -: 25.2±7.3, DOX +: 21.0±11.7, Cancer: 69.5±19.1. The gray lines represent the mean values.

Article Snippet: For CHI3L1 protein detection, BALFs from doxycycline-treated or untreated CCSP-rtTA/(TetO) 7 -CMV-Stat3 bitransgenic mice were collected as described above and diluted with Laemmli sample buffer (Bio-Rad, Hercules, CA) at 1∶1, then heated in boiling water for 5 min. Gel electrophoresis and antibody hybridization were performed following the same procedure as above except that rat anti-mouse ChI3L1 primary antibody (1∶1,000, R&D, Minneapolis, MN) and goat anti rat IgG secondary antibody (1∶3000, Vector Laboratories, Burlingame, CA) were used.

Techniques:

Journal: PLoS ONE

Article Title: Stat3 Downstream Gene Product Chitinase 3-Like 1 Is a Potential Biomarker of Inflammation-induced Lung Cancer in Multiple Mouse Lung Tumor Models and Humans

doi: 10.1371/journal.pone.0061984

Figure Lengend Snippet: CCSP-rtTA/(TetO) 7 -CMV-Stat3C bitransgenic mice were treated (+DOX) or untreated (-DOX) with doxycycline. Immunofluorescent staining of lung sections was performed using anti-CHI3L1 and F4/80 antibodies. Co-localization of both staining was observed in the merged picture. Bar represents 20 µm.

Article Snippet: For CHI3L1 protein detection, BALFs from doxycycline-treated or untreated CCSP-rtTA/(TetO) 7 -CMV-Stat3 bitransgenic mice were collected as described above and diluted with Laemmli sample buffer (Bio-Rad, Hercules, CA) at 1∶1, then heated in boiling water for 5 min. Gel electrophoresis and antibody hybridization were performed following the same procedure as above except that rat anti-mouse ChI3L1 primary antibody (1∶1,000, R&D, Minneapolis, MN) and goat anti rat IgG secondary antibody (1∶3000, Vector Laboratories, Burlingame, CA) were used.

Techniques: Staining

Journal: PLoS ONE

Article Title: Stat3 Downstream Gene Product Chitinase 3-Like 1 Is a Potential Biomarker of Inflammation-induced Lung Cancer in Multiple Mouse Lung Tumor Models and Humans

doi: 10.1371/journal.pone.0061984

Figure Lengend Snippet: A) Purity of recombinant CHI3L1-Flag fusion protein (upper arrow); B) LLC cell proliferation treated with GST or CHI3L1-GST fusion protein; C) The apoptotic activity of LLC cells treated with GST or CHI3L1-GST fusion protein by Annexin V labeling assay. *, p<0.05, **, P<0.01.

Article Snippet: For CHI3L1 protein detection, BALFs from doxycycline-treated or untreated CCSP-rtTA/(TetO) 7 -CMV-Stat3 bitransgenic mice were collected as described above and diluted with Laemmli sample buffer (Bio-Rad, Hercules, CA) at 1∶1, then heated in boiling water for 5 min. Gel electrophoresis and antibody hybridization were performed following the same procedure as above except that rat anti-mouse ChI3L1 primary antibody (1∶1,000, R&D, Minneapolis, MN) and goat anti rat IgG secondary antibody (1∶3000, Vector Laboratories, Burlingame, CA) were used.

Techniques: Recombinant, Activity Assay, Labeling

Journal: PLoS ONE

Article Title: Stat3 Downstream Gene Product Chitinase 3-Like 1 Is a Potential Biomarker of Inflammation-induced Lung Cancer in Multiple Mouse Lung Tumor Models and Humans

doi: 10.1371/journal.pone.0061984

Figure Lengend Snippet: Top: CHI3L1 protein concentrations in human serum. Bottom: ROC (Receiver Operating Characteristic) curve analysis to determine the area under the curve (AUC). Mean ± SD in control (n>30): 17.3±7.8. Mean ± SD in adenocarcinoma (n>30): 66.9±64.8. Mean ± SD in squamous carcinoma (n>30): 69.3±69.0. Mean ± SD in small cell cancer (n>30): 56.3±58.8. The gray lines represent the mean values. Control: Normal human without cancer.

Article Snippet: For CHI3L1 protein detection, BALFs from doxycycline-treated or untreated CCSP-rtTA/(TetO) 7 -CMV-Stat3 bitransgenic mice were collected as described above and diluted with Laemmli sample buffer (Bio-Rad, Hercules, CA) at 1∶1, then heated in boiling water for 5 min. Gel electrophoresis and antibody hybridization were performed following the same procedure as above except that rat anti-mouse ChI3L1 primary antibody (1∶1,000, R&D, Minneapolis, MN) and goat anti rat IgG secondary antibody (1∶3000, Vector Laboratories, Burlingame, CA) were used.

Techniques:

Journal: Nature Communications

Article Title: Regulation of chitinase-3-like-1 in T cell elicits Th1 and cytotoxic responses to inhibit lung metastasis

doi: 10.1038/s41467-017-02731-6

Figure Lengend Snippet: Chitinase and Chitinase-like protein expressions in T cells. a , b mRNA expression of chitinase (Chitotriosidase, AMCase) and chitinase-like protein (Chi3l1, Ym-1) in mouse CD4 and CD8 T cells upon anti-CD3 and anti-CD28 stimulation for 3 days. Each gene expression level was normalized to β-actin, and represented as fold change to Non-activated (NA). c , d Chi3l1 protein level in CD4 and CD8 T cells stimulated by plate-bound anti-CD3 and anti-CD28 antibodies for indicated time point. Densitometric values of band intensity were calculated by normalization to the value of β-actin. e Chi3l1 mRNA expression in naïve and effector T cell subsets. Chi3l1 mRNA expression level was normalized to β-actin level. Data are mean ± SD of three sets of independent experiments ( n = 6). n.d., not detected; ** p < 0.01, *** p < 0.001 (two-tailed Student’s t -test)

Article Snippet: Mouse anti-Chi3l1 antibody was from R&D Systems (1:1000 diluted, MAB2649), and mouse p-Erk (1:1000 diluted, #4377S), p-Akt (1:1000 diluted, #4060S), pSTAT1 (1:1000 diluted, #7649), pSTAT4 (1:1000 diluted, 4134S), pSTAT6 (1:1000 diluted, #9361 P), tSTAT1 (1:1000 diluted, #9172 P), tSTAT4 (1:1000 diluted, 2653S), and tSTAT6 (1:1000 diluted, #9362 P) antibodies and HRP-conjugated anti-rabbit (1:20000 diluted, #7074) were from Cell Signaling Technology.

Techniques: Expressing, Gene Expression, Two Tailed Test

Journal: Nature Communications

Article Title: Regulation of chitinase-3-like-1 in T cell elicits Th1 and cytotoxic responses to inhibit lung metastasis

doi: 10.1038/s41467-017-02731-6

Figure Lengend Snippet: Chi3l1 KO T cells are hyper-responsive to TcR stimulation. a , b MACS-sorted WT and Chi3l1 KO naïve CD4 T cells were activated by plate-bound anti-CD3 and anti-CD28 antibodies for 3 days. IFNγ and IL-4 expression level was analyzed by flow cytometry. c IFNγ, IL-2, IL-4, and IL-17 cytokine production in the CD4 T cell culture supernatant was measured by ELISA. d IFNγ and TNFα cytokine production in the CD8 T cell culture supernatant was measured by ELISA. e Proportion of proliferating CD4 T cells were analyzed by CFSE assay. f Percentages of proliferating and non-proliferating cells were analyzed by gating on histogram. g Phosphorylated Erk, Akt, STAT1, STAT5 were analyzed by Western blotting. h Relative densitometric analysis of Western blots was represented as normalized to β-actin. Data are mean ± SD of three sets of independent experiments ( n = 6). n.s., not significant; * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed Student’s t -test)

Article Snippet: Mouse anti-Chi3l1 antibody was from R&D Systems (1:1000 diluted, MAB2649), and mouse p-Erk (1:1000 diluted, #4377S), p-Akt (1:1000 diluted, #4060S), pSTAT1 (1:1000 diluted, #7649), pSTAT4 (1:1000 diluted, 4134S), pSTAT6 (1:1000 diluted, #9361 P), tSTAT1 (1:1000 diluted, #9172 P), tSTAT4 (1:1000 diluted, 2653S), and tSTAT6 (1:1000 diluted, #9362 P) antibodies and HRP-conjugated anti-rabbit (1:20000 diluted, #7074) were from Cell Signaling Technology.

Techniques: Expressing, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, CFSE Assay, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Regulation of chitinase-3-like-1 in T cell elicits Th1 and cytotoxic responses to inhibit lung metastasis

doi: 10.1038/s41467-017-02731-6

Figure Lengend Snippet: Chi3l1 KO CD4 T cells are prone to Th1 differentiation. a , b MACS-sorted WT and Chi3l1 KO naive CD4 T cells were differentiated into Th1, Th2, and Th17 cells under specific cytokine-skewing condition, and Intracellular cytokine staining performed to analyze lineage-specific cytokine expression. c IFNγ, TNFα, IL-4, and IL-17 production in culture supernatants were measured by ELISA. d Lineage specific mRNA levels were analyzed by quantitative RT-PCR. Each gene expression level was normalized to β-actin, and represented as relative expression to WT. Data are mean ± SD of eight sets of independent experiments ( n = 16). n.s., not significant; * p < 0.05, ** p < 0.01 (two-tailed Student’s t -test)

Article Snippet: Mouse anti-Chi3l1 antibody was from R&D Systems (1:1000 diluted, MAB2649), and mouse p-Erk (1:1000 diluted, #4377S), p-Akt (1:1000 diluted, #4060S), pSTAT1 (1:1000 diluted, #7649), pSTAT4 (1:1000 diluted, 4134S), pSTAT6 (1:1000 diluted, #9361 P), tSTAT1 (1:1000 diluted, #9172 P), tSTAT4 (1:1000 diluted, 2653S), and tSTAT6 (1:1000 diluted, #9362 P) antibodies and HRP-conjugated anti-rabbit (1:20000 diluted, #7074) were from Cell Signaling Technology.

Techniques: Staining, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Gene Expression, Two Tailed Test

Journal: Nature Communications

Article Title: Regulation of chitinase-3-like-1 in T cell elicits Th1 and cytotoxic responses to inhibit lung metastasis

doi: 10.1038/s41467-017-02731-6

Figure Lengend Snippet: Increased Th1 and CTL response in Chi3l1 KO T cells are IFNγ dependent. a Phosphorylation of STAT1, STAT4, STAT6 were analyzed by Western blotting. Relative densitometric analysis of Western blotting normalized to total STAT. b WT and Chi3l1 KO naive CD4 T cells were differentiated into Th1 cells with the indicated concentrations of IFNγ neutralizing antibodies and assessed for IFNγ and IL-4 expression by intracellular cytokine staining. c Proportion of IFNγ producing cells were represented by relative % of IFNγ to WT Th1. d FACS-sorted WT and Chi3l1 KO naïve CD4 T cells were treated with IFNγ and pSTAT1 level was analyzed by Western blotting. Densitometric values of band intensity was calculated by normalization to the value of β-actin. e Intracellular level of IFNγ and Granzyme B in CD8 T cells. f Proportion of IFNγ producing cells were represented as relative % of IFNγ to WT CD8 T cells without anti-IFNγ neutralizing antibody. g Proportion of Granzyme B expressing cells were represented as relative % of Granzyme B to WT CD8 T cells without anti-IFNγ neutralizing antibody. Data are mean ± SD of three sets of independent experiments ( n = 6). n.s., not significant; * p < 0.05, *** p < 0.001

Article Snippet: Mouse anti-Chi3l1 antibody was from R&D Systems (1:1000 diluted, MAB2649), and mouse p-Erk (1:1000 diluted, #4377S), p-Akt (1:1000 diluted, #4060S), pSTAT1 (1:1000 diluted, #7649), pSTAT4 (1:1000 diluted, 4134S), pSTAT6 (1:1000 diluted, #9361 P), tSTAT1 (1:1000 diluted, #9172 P), tSTAT4 (1:1000 diluted, 2653S), and tSTAT6 (1:1000 diluted, #9362 P) antibodies and HRP-conjugated anti-rabbit (1:20000 diluted, #7074) were from Cell Signaling Technology.

Techniques: Phospho-proteomics, Western Blot, Expressing, Staining

Journal: Nature Communications

Article Title: Regulation of chitinase-3-like-1 in T cell elicits Th1 and cytotoxic responses to inhibit lung metastasis

doi: 10.1038/s41467-017-02731-6

Figure Lengend Snippet: RNA transcriptomes of Chi3l1 KO T cells. a 100-bp pair-ended RNA-sequencing in naïve, Th1-skewed WT, and Chi3l1 KO CD4 T cells. The number of up-regulated genes were indicated as red, and down-regulated genes were indicated as green in each comparative analysis. b Heatmap analysis of genes of interest. c Scatterplot indicates either of over than two-fold up-regulated and down-regulated genes in WT Th1 versus Chi3l1 KO Th1 comparison. d Expression of genes of interest related to cytotoxicity and the IFNγ signaling pathway in Th1 cells were confirmed by quantitative RT-PCR. Each gene expression level was normalized to β-actin, and represented as relative expression to WT. e Quantitative RT-PCR was performed in activated CD8 T cells. Each gene expression level was normalized to β-actin, and represented as relative to WT. Data are mean ± SD of ten sets of independent experiments ( n = 20). n.s., not significant; * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed Student’s t -test)

Article Snippet: Mouse anti-Chi3l1 antibody was from R&D Systems (1:1000 diluted, MAB2649), and mouse p-Erk (1:1000 diluted, #4377S), p-Akt (1:1000 diluted, #4060S), pSTAT1 (1:1000 diluted, #7649), pSTAT4 (1:1000 diluted, 4134S), pSTAT6 (1:1000 diluted, #9361 P), tSTAT1 (1:1000 diluted, #9172 P), tSTAT4 (1:1000 diluted, 2653S), and tSTAT6 (1:1000 diluted, #9362 P) antibodies and HRP-conjugated anti-rabbit (1:20000 diluted, #7074) were from Cell Signaling Technology.

Techniques: RNA Sequencing, Comparison, Expressing, Quantitative RT-PCR, Gene Expression, Two Tailed Test

Journal: Nature Communications

Article Title: Regulation of chitinase-3-like-1 in T cell elicits Th1 and cytotoxic responses to inhibit lung metastasis

doi: 10.1038/s41467-017-02731-6

Figure Lengend Snippet: Chi3l1 KO mice have reduced pulmonary metastasis with increased IFNγ-producing CD4 and CD8 T cells in the lung. a Representative lung image from B16F10 melanoma injected WT and Chi3l1 KO mice. Scale bar, 2 mm b Number of pleural colonies in each lung was counted. Data are mean ± SEM of three sets of independent experiments and each dot in graphs represent an individual mouse. c Relative total tumor area in the lung was measured by Image J software 1.48 v. d Histology of lung sections by H&E staining, and infiltrated tumor region was measured by Image J software 1.48 v. Scale bar, 200 m. e , f IFNγ producing CD4 T cells, Foxp3 + regulatory T cells, IFNγ producing CD8 T cells, and Granzyme B expression in IFNγ + CD8 T cells in the lung was analyzed by intracellular cytokine staining. % of IFNγ + , % of Foxp3 + , and MFI of Granzyme B was represented as scattered graph. g , h IFNγ producing NK cells IFNγ producing non-lymphocytic population, and Granzyme B expression in IFNγ + NK cells and non-lymphocytic population in the lung was analyzed. % of IFNγ + , and MFI of Granzyme B was represented as scattered graph. i mRNA expression of genes related to cytotoxicity and Th1 effector functions was analyzed by quantitative RT-PCR. Each gene expression level was normalized to β-actin. j Cytotoxicity of WT and Chi3l1 KO CD8 T cells against B16F10 target cells at indicated E:T ratios. k NK cell activity was represented as tumor killing activity of WT and Chi3l1 KO NK cells against to B16F10 target cells at indicated E:T ratios. Data are mean ± SD of three sets of independent experiments and each dot in graphs represent an individual mouse. n.s., not significant; * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed Student’s t -test)

Article Snippet: Mouse anti-Chi3l1 antibody was from R&D Systems (1:1000 diluted, MAB2649), and mouse p-Erk (1:1000 diluted, #4377S), p-Akt (1:1000 diluted, #4060S), pSTAT1 (1:1000 diluted, #7649), pSTAT4 (1:1000 diluted, 4134S), pSTAT6 (1:1000 diluted, #9361 P), tSTAT1 (1:1000 diluted, #9172 P), tSTAT4 (1:1000 diluted, 2653S), and tSTAT6 (1:1000 diluted, #9362 P) antibodies and HRP-conjugated anti-rabbit (1:20000 diluted, #7074) were from Cell Signaling Technology.

Techniques: Injection, Software, Staining, Expressing, Quantitative RT-PCR, Gene Expression, Activity Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Regulation of chitinase-3-like-1 in T cell elicits Th1 and cytotoxic responses to inhibit lung metastasis

doi: 10.1038/s41467-017-02731-6

Figure Lengend Snippet: Chi3l1 deletion in T cell inhibits pulmonary metastasis with increase of Th1 and CTL. a Genotyping PCR to confirm CD4 specific deletion of Chi3l1 in the mice. b Representative lung image from B16F10 melanoma injected WT and CD4-Chi3l1 KO mice. Scale bar, 2 mm c Number of pleural colonies in each lung was counted. Data are mean ± SEM of three sets of independent experiments and each dot in graphs represent an individual mouse. d Histology of lung sections by H&E staining. Scale bar, 200 m ( e , f ) IFNγ and/or TNFα producing CD4 T cells and CD8 T cells in the lung was analyzed by intracellular cytokine staining. % of IFNγ + , % of TNFα + , and % of TNFα + IFNγ + was represented as scattered graph. g , h IFNγ and/or TNFα producing NK cells, and non-lymphocytic populations (NK1.1 - CD4 - CD8 - ) in the lung was analyzed by intracellular cytokine staining. % of IFNγ + , % of TNFα + , and % of TNFα + IFNγ + was represented as scattered graph. i mRNA expression of genes related to cytotoxicity and Th1 effector functions was analyzed by quantitative RT-PCR. Each gene expression level was normalized to β-actin. Data are mean ± SD of three sets of independent experiments and each dot in graphs represent an individual mouse. n.s., not significant; * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed Student’s t -test)

Article Snippet: Mouse anti-Chi3l1 antibody was from R&D Systems (1:1000 diluted, MAB2649), and mouse p-Erk (1:1000 diluted, #4377S), p-Akt (1:1000 diluted, #4060S), pSTAT1 (1:1000 diluted, #7649), pSTAT4 (1:1000 diluted, 4134S), pSTAT6 (1:1000 diluted, #9361 P), tSTAT1 (1:1000 diluted, #9172 P), tSTAT4 (1:1000 diluted, 2653S), and tSTAT6 (1:1000 diluted, #9362 P) antibodies and HRP-conjugated anti-rabbit (1:20000 diluted, #7074) were from Cell Signaling Technology.

Techniques: Injection, Staining, Expressing, Quantitative RT-PCR, Gene Expression, Two Tailed Test

Journal: Nature Communications

Article Title: Regulation of chitinase-3-like-1 in T cell elicits Th1 and cytotoxic responses to inhibit lung metastasis

doi: 10.1038/s41467-017-02731-6

Figure Lengend Snippet: Targeted inhibition of Chi3l1 by dNP2-siChi3l1 complex enhances effector cytokine production in Th1 and CD8 T cells. a Schematic diagram of the complex formation of dNP2-HA2 peptide and Chi3l1 siRNA (siChi3l1) at indicated N/P ratios. b Gel retardation assay of dNP2-siChi3l1 complex. c The size of free siRNA, free peptide, and dNP2-siRNA complexes were measured by Nano particle size analyzer. d Dose dependent reduction of Chi3l1 mRNA expression by dNP2-siChi3l1 complex at 1:25 N/P ratio. e Chi3l1 protein level in culture supernatants were measured by ELISA. f Chi3l1 mRNA in the lung after intranasal administration of dNP2-siEGFP or dNP2-siChi3l1 complex at 1:25 N/P ratio. Chi3l1 mRNA normalized to β-actin, and presented as relative to expression at day 0. g Chi3l1 protein level in the lung was analyzed by Western blotting. Chi3l1 protein relative to expression at day 0. h Densitometric values of band intensity was calculated by normalization to the value of β-actin, then the value was represented as relative to the value at day 0. Statistical significance of dNP2-siChi3l1 treated group was analyzed to dNP2-siEGFP treated group on each day. i FACS-sorted WT naïve CD4 T cells were differentiated into Th1 cells and WT naïve CD8 T cells were activated by plate-bound anti-CD3 and anti-CD28 antibodies with IL-2 for 5 days with indicated concentrations of dNP2-siEGFP or dNP2-siChi3l1 complexes. IFNγ and TNFα expression were analyzed by flow cytometry. j % of TNFα + IFNγ + cells were represented as bar graph. k Chi3l1 and IFNγ mRNA expression in dNP2-siChi3l1 treated Th1 cells. Each gene expression level was normalized to β-actin, and represented as fold change to those of WT Th1 cell. Data are presented as mean ± SD of three sets of independent experiments ( n = 6). n.s., not significant; * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed Student’s t -test)

Article Snippet: Mouse anti-Chi3l1 antibody was from R&D Systems (1:1000 diluted, MAB2649), and mouse p-Erk (1:1000 diluted, #4377S), p-Akt (1:1000 diluted, #4060S), pSTAT1 (1:1000 diluted, #7649), pSTAT4 (1:1000 diluted, 4134S), pSTAT6 (1:1000 diluted, #9361 P), tSTAT1 (1:1000 diluted, #9172 P), tSTAT4 (1:1000 diluted, 2653S), and tSTAT6 (1:1000 diluted, #9362 P) antibodies and HRP-conjugated anti-rabbit (1:20000 diluted, #7074) were from Cell Signaling Technology.

Techniques: Inhibition, Electrophoretic Mobility Shift Assay, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Flow Cytometry, Gene Expression, Two Tailed Test