Journal: Scientific Reports

Article Title: Divergent roles for Clusterin in Lung Injury and Repair

doi: 10.1038/s41598-017-15670-5

Figure Lengend Snippet: Elevated extracellular and reduced cell associated Clusterin in Idiopathic Pulmonary Fibrosis. ( A ) Clusterin gene expression was quantitated using RT-PCR in lung tissue from healthy control lung tissue (n = 10), COPD patients (n = 19) and IPF patients (n = 54). ( B,C ) Circulating Clusterin protein levels were quantitated and compared between IPF (n = 60) and a cohort of age matched controls (n = 30) ( B ), and from COPD (n = 15) and a separate cohort of age matched controls (n = 25) ( C ). Levels were measured by Somascan analysis, each dot representing a different individual. ( D–J ) Clusterin expression was visualized (brown staining) by IHC analysis of three IPF lungs ( D – I ) and a representative normal lung ( J ) tissue, size bars are indicated on image. ( K ) The staining intensity of cell-associated Clusterin was quantified in airway epithelial cells using Aperio Scanscope software. Shown is the average Clusterin staining intensity in airway epithelial cells in normal, IPF and COPD lung tissue. Data are expressed as Mean ± SEM *P ≤ 0.05, ****P ≤ 0.001 significance to relevant control levels.

Article Snippet: After blocking, tissues were stained with goat anti-mouse Clusterin (R&D Systems), mouse anti human Clusterin (BD Pharmingen) diluted in PBST, FITC conjugated anti-8-hydroxyguanosine (Abcam), p-H2A.X (Cell Signaling) or rabbit anti-cleaved Caspase-3 (Cell Signaling) antibodies with background reducing components (DAKO) for 1 hour at room temperature.

Techniques: Gene Expression, Reverse Transcription Polymerase Chain Reaction, Control, Expressing, Staining, Software

Journal: Scientific Reports

Article Title: Divergent roles for Clusterin in Lung Injury and Repair

doi: 10.1038/s41598-017-15670-5

Figure Lengend Snippet: Induction of Clusterin in response to bleomycin-induced lung injury. ( A–H ) C57Bl/6 mice were given bleomycin intratracheally and CLU expression was visualized by IHC after 7 ( C , D ), 14 ( E , F ) and 21 ( G , H ) days of bleomycin instillation and compared to staining in the lungs of Day 21 saline treated mice ( A , B ). ( I ) Clusterin transcript expression was quantitated after 14, 21 and 28 days of bleomycin instillation by RT-PCR in whole lung samples. Data are expressed as Mean ± SEM*P ≤ 0.05, **P ≤ 0.01 significance to relevant control levels. ( J–Q ) Saline or bleomycin challenged C57Bl/6 mice were treated with 20 µg of recombinant Clusterin every three days starting at Day 2. Shown are representative histological images of the lungs stained with Masson’s trichrome from saline + PBS vehicle ( J , K ), saline + Clusterin ( L , M ), bleomycin + PBS ( N , O ) and bleomycin + Clusterin ( P , Q ) treated mice. The top images are of whole scans of the lungs and the bottom images are taken at 200x magnification. ( R ) Collagen content in the lungs of the recombinant Clusterin and vehicle treated mice was biochemically quantified using a hydroxyproline assay. Shown is the average hydroxyproline (µg) from right lung lobes. ( S , T ) Shown is the average total BAL cell number ( S ) and the total TGFß levels ( T ) in the lungs of vehicle or Clusterin treated mice 15 days after bleomycin challenge. ( U ) FN1, VEGFA, PDGFRA and MMP12 transcript expression was quantified in whole lung samples 15 days after bleomycin, vehicle and/or Clusterin treatment. Shown is the average expression from 3 Saline/PBS, 3 Saline/rClusterin, 7 Bleo/PBS and 7 Bleo/rClusterin treated mice.

Article Snippet: After blocking, tissues were stained with goat anti-mouse Clusterin (R&D Systems), mouse anti human Clusterin (BD Pharmingen) diluted in PBST, FITC conjugated anti-8-hydroxyguanosine (Abcam), p-H2A.X (Cell Signaling) or rabbit anti-cleaved Caspase-3 (Cell Signaling) antibodies with background reducing components (DAKO) for 1 hour at room temperature.

Techniques: Expressing, Staining, Saline, Reverse Transcription Polymerase Chain Reaction, Control, Recombinant, Hydroxyproline Assay

Journal: Scientific Reports

Article Title: Divergent roles for Clusterin in Lung Injury and Repair

doi: 10.1038/s41598-017-15670-5

Figure Lengend Snippet: Clusterin deficient mice show persistent fibrosis in response to bleomycin. WT or CLU −/− received bleomycin intratracheally on Day 0. ( A–H ) Depicted is Masson’s Trichrome histological staining of WT saline ( A , B ), WT bleomycin ( C , D ), CLU−/− saline ( E,F ) and CLU−/− bleomycin ( G , H ) treated lungs fourteen days after instillation. Shown are whole lung images (top) and magnified images (bottom). ( I ) Collagen content in the lungs of the of bleomycin treated wildtype and CLU−/− mice was biochemically quantified using a hydroxyproline assay, 14 days after bleomycin instillation. Shown is the average hydroxyproline (µg) from right lung lobes. ( J ) Shown is the average total TGFβ levels in the BAL from wildtype and CLU−/− mice, 14 days after bleomycin challenge. ( K ) Shown is the average IL-1β levels in whole lung lysates from wildtype and CLU−/− mice, 14 days after bleomycin challenge. ( L , M ) The proliferative status ( L ) and Caspase3 expression ( M ) in live CD45 − EpCAM + epithelial cells was assessed via Ki67 and activated Caspase-3 staining, respectively, 5 days after bleomycin challenge. Depicted is the fold change in cell number in response to bleomycin, relative to its relevant saline control. ( N , O ) IHC analysis for cleaved Caspase3 was performed on fibrotic murine lungs and quantified using Aperio Scanscope software. Depicted is the fold change in clusters, as defined by three or more adjacent positive cells, staining for cleaved Caspase3, 14 ( N ) and 28 ( O ) days after bleomycin challenge, compared to relevant saline control. ( P – W ) Depicted is Masson’s Trichrome histological staining of WT saline ( P , Q ), WT bleomycin ( R , S ), CLU−/− saline ( T,U ) and CLU−/− bleomycin ( V , W ) treated lungs 28 days after instillation. Shown are whole lung images (top) and magnified images (bottom). ( X ) Collagen content in the lungs of the of wildtype and CLU−/− treated mice was biochemically quantified using a hydroxyproline assay 28 days after bleomycin instillation. Shown is the average hydroxyproline (µg) from right lung lobes. ( Y , Z ) Quantitative PCR analysis was performed on RNA purified from lungs of 28-day saline and bleomycin treated murine lungs. Shown is the average col3a1 ( Y ) and fn1 ( Z ) transcript expression. Data are Mean ± SEM., n = 8–13 mice/ group. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.005, ****P ≤ 0.001 significance, or as stated. ns = not significant.

Article Snippet: After blocking, tissues were stained with goat anti-mouse Clusterin (R&D Systems), mouse anti human Clusterin (BD Pharmingen) diluted in PBST, FITC conjugated anti-8-hydroxyguanosine (Abcam), p-H2A.X (Cell Signaling) or rabbit anti-cleaved Caspase-3 (Cell Signaling) antibodies with background reducing components (DAKO) for 1 hour at room temperature.

Techniques: Staining, Saline, Hydroxyproline Assay, Expressing, Control, Software, Real-time Polymerase Chain Reaction, Purification

Journal: Scientific Reports

Article Title: Divergent roles for Clusterin in Lung Injury and Repair

doi: 10.1038/s41598-017-15670-5

Figure Lengend Snippet: Loss of transcripts for Clusterin, MMR, BER & DSB DNA repair pathway components in IPF SSEA4 + basal-like epithelial cells. ( A ) SSEA4 + and SSEA4 − cells were sorted from normal and IPF stromal cultures, RNA was extracted and subjected to RNAseq analysis. Fold changes in IPF relative to normal were calculated and the results were uploaded onto ingenuity IPA for pathway analysis ( A ). ( B ) Shown is an representative sort plot depicting the forward and side scatter localization of SSEA4 + cells. ( C ) Normalized RNA sequencing FPKM values were mined for transcripts that were enriched in SSEA4 + relative to SSEA4 − cells. Shown are enriched basal cell lineage markers in normal lung and IPF SSEA4 + cells. ( D ) Shown is the average CLU transcript FPKM values normal and IPF SSEA4 + cells. ( E–G ) Transcripts encoding for components of MMR ( E ), BER ( F ) and DSB ( G ) pathways were mined from SSEA4 + RNAseq data, clustered using Euclidean distance with complete linkage and heat maps were generated using Morpheus (Broad Institute). ( H–M ) Normal and IPF lung biopsies were stained with anti-SSEA4 and BRCA1 antibodies followed by fluorescent microscopic analysis. Representative images were taken at 200x magnification from two normal and four IPF lung biopsies stained with BRCA1 ( H , K respectively), SSEA4 ( I , L respectively) and the merged composites ( J , M respectively). ( N ) The number of SSEA4 + cells showing both SSEA4 and BRCA1 staining was quantified, averaged, and the percentage of cells was calculated from 28 normal and 84 IPF SSEA4 + cells from 3 normal and 4 IPF lungs, respectively.

Article Snippet: After blocking, tissues were stained with goat anti-mouse Clusterin (R&D Systems), mouse anti human Clusterin (BD Pharmingen) diluted in PBST, FITC conjugated anti-8-hydroxyguanosine (Abcam), p-H2A.X (Cell Signaling) or rabbit anti-cleaved Caspase-3 (Cell Signaling) antibodies with background reducing components (DAKO) for 1 hour at room temperature.

Techniques: RNA Sequencing, Generated, Staining

Journal: Scientific Reports

Article Title: Divergent roles for Clusterin in Lung Injury and Repair

doi: 10.1038/s41598-017-15670-5

Figure Lengend Snippet: Pictorial summary for the potential role of Clusterin variants in the modulation of epithelial cell survival and lung fibrosis.

Article Snippet: After blocking, tissues were stained with goat anti-mouse Clusterin (R&D Systems), mouse anti human Clusterin (BD Pharmingen) diluted in PBST, FITC conjugated anti-8-hydroxyguanosine (Abcam), p-H2A.X (Cell Signaling) or rabbit anti-cleaved Caspase-3 (Cell Signaling) antibodies with background reducing components (DAKO) for 1 hour at room temperature.

Techniques:

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Immunoregulatory properties of cell free DNA

doi: 10.1007/s00018-025-05862-y

Figure Lengend Snippet: cfDNA-induced variation in gene expression. ( A ) 40 genes analyzed by Bio-Rad iQ5 analysis in LPS pre-activated PMJ2-PC macrophages treated (blue bar) or not (green bar) with the poly-C/poly-G oligonucleotide (1 µg/ml for 3 h), * p -value < 0.05 and ** p -value < 0.001; ( B ) Quantitative PCR analysis on mRNAs coding for MAPK-14 , EGR-1 , and CCL22 in LPS pre-activated PMJ2-PC macrophages treated (black bar) or not (white bar) with the poly-C/poly-G oligonucleotide (1 µg/ml for 3 h). Data are expressed as fold change (2 − ΔΔCT ) normalized with GAPDH. Data are the mean of 3 concordant experiments; Student paired T test ( C ) Network analysis using the STRING-DB software of differentially expressed genes ( EGR-1 and CCL22 ) between pre-activated PMJ2-PC macrophages treated or not with the poly-C/poly-G oligonucleotide. EGR-1 and CCL22 genes are showed in red circles

Article Snippet: A pre-designed panel of pathway-specific genes (Supplementary Table ) was used to test the expression of 40 genes with Bio-Rad iQ5 real-time PCR system (Biorad, Hercules, CA) and analyzed by PrimePCR analysis Software Gene Study 1.0.030.1023.

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Software

Journal: iScience

Article Title: Merkel cell polyomavirus T-antigens regulate DICER1 mRNA stability and translation through HSC70

doi: 10.1016/j.isci.2021.103264

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-MYC antibody (9E10) , Santa Cruz Biotechnology , sc-40 RRID: AB_2857941.

Techniques: Virus, Recombinant, Transfection, Electroporation, Bicinchoninic Acid Protein Assay, Reverse Transcription, Gene Expression, Polymerase Chain Reaction, Luciferase, Mutagenesis, In Vitro, Construct, shRNA, Software

Journal: Journal for Immunotherapy of Cancer

Article Title: Extracellular HMGB1 blockade inhibits tumor growth through profoundly remodeling immune microenvironment and enhances checkpoint inhibitor-based immunotherapy

doi: 10.1136/jitc-2020-001966

Figure Lengend Snippet: HMGB1 is highly secreted by basal-like breast cancer cells and its tumor-specific cytoplasmic expression is associated with immune tolerance and poor outcome. (A) The METABRIC dataset was used for analyzing HMGB1 expression level in breast cancers according to molecular subtypes, histologic grades, nodal and metastatic statuses. (B) Representative pictures of normal mammary glands and breast cancers stained for HMGB1. Note the two distinct HMGB1 staining patterns detected in tumor specimens: nuclear versus cytoplasmic. (C) Semiquantitative evaluation of cytoplasmic HMGB1 immunoreactivity (negative, 0%–10% or >10% positive cells) in both normal mammary glands (n=120) and neoplasms (LumA, n=35; LumB, n=31; HER2+, n=37; basal-like, n=172). (D) Disease-free survival of patients treated for basal-like breast cancer according to cytoplasmic HMGB1 expression (negative, n=72; 0%–10%, n=61; >10%, n=39). This latter parameter was clearly found to be an independent prognostic factor. (E) Illustration of the different steps for DAB-positive cell quantification using computerized image analysis (QuPath). (F) CD3 + , Foxp3 + , CD68 + and CD206 + cell infiltrations in microenvironment of basal-like breast tumors. Whereas the global number (CD68 + ) did not significantly change, an increased density of CD206 + M2 macrophages was detected in cytoplasmic HMGB1-positive cancers. A similar increase was also reported with Foxp3 + Treg lymphocytes. the number of positive cells was reported to tumor area (mm 2 ). (G) Representative examples of normal (HMEC and MCF10A) and malignant cells (LumA: T-47D and MCF7; basal-like: MDA-MB-468 and Hs578T) stained for HMGB1. Note the exclusive nuclear immunoreactivity displayed by normal mammary cells. Secretion/release of HMGB1 analyzed by ELISA in (H) human and (I) mouse cell culture supernatants. High concentrations were especially detected in cell cultures derived from triple negative/basal-like tumors. The means±SEM (plus each individual data point) for at least three independent experiments are represented. The scale bar represents 100 µm. Asterisks indicate statistically significant differences (*p<0.05; **p<0.01; ***p<0.001). P values were determined using one-way ANOVA followed by Bonferroni post-test (A), unpaired t-test (A), χ 2 test (C), log-rank (Mantel-Cox) test (D) and one-way ANOVA followed by Dunnett’s multiple comparison post-test (F, H, I). ANOVA, analysis of variance; HMGB1, high-mobility group box 1; METABRIC, Molecular Taxonomy of Breast Cancer International Consortium.

Article Snippet: Cell culture supernatant was then harvested and HMGB1 release by both human and mouse breast cancer cells was quantified by ELISA using the following commercially available kit: HMGB1 Detection kit (Chondrex).

Techniques: Expressing, Staining, Enzyme-linked Immunosorbent Assay, Cell Culture, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Extracellular HMGB1 blockade inhibits tumor growth through profoundly remodeling immune microenvironment and enhances checkpoint inhibitor-based immunotherapy

doi: 10.1136/jitc-2020-001966

Figure Lengend Snippet: Glycyrrhizin, RAP, A box and EP exert efficient neutralizing effects on extracellular HMGB1 without altering tumor cell proliferation and apoptosis/necrosis. (A) Schematic representation of different modes of inhibition for extracellular HMGB1. Mouse RAW 264.7 cells were stimulated with recombinant HMGB1 (B) or conditioned media from 4T1 basal-like breast cancer cells (C) in the absence or presence of glycyrrhizin, RAP or a box (several concentrations were tested). Note the significant decrease of HMGB1-induced TNFα secretion when HMGB1 inhibitors were added in the cell cultures, indicating their efficient neutralizing effect. (D) EP was directly added in the culture medium of 4 different mouse basal-like breast cancer cell lines (4T1, 67NR, EpRas and EpH4). Forty-eight hours later, HMGB1 concentrations were determined by ELISA and the ability of EP to inhibit HMGB1 release in a dose-dependent manner was highlighted. (E) Oxygen consumption rate (OCR) and (F) extracellular acidification rate (ECAR) in 4T1 cells in the absence or presence of glycyrrhizin (1 nM), RAP (10 µM) and a box (0.5 µg/mL) were determined using Seahorse extracellular flux analyzer. No modification of OCR/ECAR was detected with these three HMGB1 inhibitors. (G) OCR and (H) ECAR in 4T1 cells following EP addition (concentration range: 0.1–10 mM). Histograms representing OCR (I) and ECAR (J) before (baseline) and after (stressed) oligomycin and FCCP addition in the absence or presence of EP. Both OCR and ECAR were strongly decreased with 5 and 10 mM EP. No significant change was detected with lower concentrations (0.1–1 mM). (K) Cell proliferation and (L) apoptosis of mouse 4T1 cells cultured without or with HMGB1 inhibitors (glycyrrhizin (1 nM), RAP (10 µM), a box (0.5 µg/mL) and EP (1 mM)) were determined using IncuCyte live cell analyzing system and annexin V-propidium iodide staining assay, respectively. No significant change was reported. The means±SEM (plus each individual data point) for at least three independent experiments are represented. Asterisks indicate statistically significant differences (*p<0.05; **p<0.01; ***p<0.001). P values were determined using one-way ANOVA, followed by Dunnett’s multiple comparison post-test (B, C, D, I, J, K, L). ANOVA, analysis of variance; ECAR, extracellular acidification rate; EP, ethyl pyruvate; HMGB1, high-mobility group box 1; RAGE, receptor for advanced glycation endproducts; RAP, RAGE antagonist peptide; TNFα, tumor necrosis factor-α.

Article Snippet: Cell culture supernatant was then harvested and HMGB1 release by both human and mouse breast cancer cells was quantified by ELISA using the following commercially available kit: HMGB1 Detection kit (Chondrex).

Techniques: Inhibition, Recombinant, Enzyme-linked Immunosorbent Assay, Modification, Concentration Assay, Cell Culture, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: Extracellular HMGB1 blockade inhibits tumor growth through profoundly remodeling immune microenvironment and enhances checkpoint inhibitor-based immunotherapy

doi: 10.1136/jitc-2020-001966

Figure Lengend Snippet: Extracellular HMGB1 blockade inhibits the growth of pre-established solid tumors in immunocompetent mice through activating anticancer immune responses. (A) Mouse basal-like breast cancer cells (4T1, 67NR and EpRas) were orthotopically injected into the mammary fat pad of immunocompetent BALB/c mice. Tumor-bearing mice were then treated at 3-day intervals with PBS (control) or HMGB1 inhibitors (glycyrrhizin (1 nM/kg), RAP (10 µM/kg), a box (500 µg/kg) and EP (1 mM/kg)). The mean tumor volumes±SEM are represented. (B) HMGB1 inhibitors were tested in nude mice implanted with 67NR cells. Note the absence of beneficial effect in these latter immunocompromised mice, indicating the dependence on the adaptive immune responses. (C) At day 17, 19 or 20 (depending on the analyzed cell line), tumors were harvested, CD45 + immune cells were isolated and analyzed by flow cytometry. The proportions of each analyzed immune cell population in both control and treated groups (pooled results) are shown. Note the drastic reduction of MDSC following extracellular HMGB1 blockade. (D) Total number of (CD45 + ) immune cells per milligram of tumor in both control and treated groups. (E) Scatter dot plots showing the percentage of each individual immune cell population (DC, PDC, CD4 + and CD8 + T cells, monocytic and granulocytic MDSC, neutrophils, M1 and M2 macrophages) among CD45 + cells in the different treatment groups. an increased M1/M2 ratio of macrophages was observed in most HMGB1 inhibitor-treated tumors. The intratumoral immune cells were analyzed in five mice per condition. (F) Scatter dot plots illustrating the percentage of tumor-infiltrating Treg (Foxp3 + ) CD4 + and CD8 + cells among total CD4 + and CD8 + populations in the different treatment groups. (G) Scatter dot plots illustrating the percentage of tumor-infiltrating PD-1 + CD4 + and PD-1 + CD8 + cells among total CD4 + and CD8 + populations in the treatment groups. The activation status of both DC (H) and PDC (I) in the different treatment groups was also determined by analyzing the expression of several cell surface markers (CD80, CD86, I-A/I-E, ILT3 and ICOSL). Data represent the mean fluorescent intensity (MFI)±SEM of 5 independent experiments in each group (each individual data point is shown). The number of apoptotic cancer cells (cleaved caspase 3 + ) (J) as well as the density of blood vessels within tumor microenvironment (CD31 + ) (K) were determined by computerized counting (using QuPath software). The number of cleaved caspase 3 + cells and the percentage of CD31 + pixels were reported to tumor area. The scale bar represents 100 µm. Asterisks indicate statistically significant differences (*p<0.05; **p<0.01; ***p<0.001). P values were determined using one-way ANOVA followed by Dunnett’s multiple comparison post-test (A, B, D, E, F, G, H, I) and (Welch-corrected) unpaired t-test (J, K). ANOVA, analysis of variance; DC, dendritic cell; EP, ethyl pyruvate; HMGB1, high-mobility group box 1; i.p, intraperitoneal; MDSC, myeloid-derived suppressor cells; pDC, plasmacytoid DC; RAP, RAGE antagonist peptide.

Article Snippet: Cell culture supernatant was then harvested and HMGB1 release by both human and mouse breast cancer cells was quantified by ELISA using the following commercially available kit: HMGB1 Detection kit (Chondrex).

Techniques: Injection, Isolation, Flow Cytometry, Activation Assay, Expressing, Software, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Extracellular HMGB1 blockade inhibits tumor growth through profoundly remodeling immune microenvironment and enhances checkpoint inhibitor-based immunotherapy

doi: 10.1136/jitc-2020-001966

Figure Lengend Snippet: Combination of anti-PD-L1 with HMGB1 inhibitors strongly inhibits tumor growth in syngeneic mouse models of basal-like breast cancer. (A) mRNA level of PD-L1 ( CD274 gene) in the four major molecular subtypes of breast cancer was determined using the METABRIC public dataset. (B) Representative example of breast cancer stained for PD-L1. Positive signals were detected on cancer cells and/or on inflammatory cells within tumor microenvironment. Semiquantitative evaluation of PD-L1 immunoreactivity (negative or >1% membrane staining) displayed by cancer cells (C) or inflammatory cells infiltrating the tumor microenvironment (D). The analyzed cancer specimens were categorized into the four molecular subtypes of breast cancer (LumA, LumB, HER2 + and basal-like). (E) The percentage of PD-L1 + cells in both epithelial/cancer (CD45 - ) and inflammatory (CD45 + ) components of untreated harvested 4T1/67NR tumors was determined by flow cytometry. Note the distinct profile displayed by these two cell lines. (F) Mouse breast cancer cells (4T1 and 67NR) were orthotopically injected into the mammary fat pad of immunocompetent BALB/c mice. Anti-PD-L1 antibody was tested alone (i.p. injection of 100 µg at days 4, 7 and 11) and in combination with HMGB1 inhibitors (RAP (10 µM/kg) and EP (1 mM/kg), treatment at 3-day intervals). The mean tumor volumes±SEM are represented. (G) The total number of (CD45 + ) immune cells per milligram of tumor was determined in the different treatment groups by flow cytometry. (H) Scatter dot plots illustrating the percentage of each individual immune cell population (DC, PDC, CD4 + and CD8 + T cells, monocytic and granulocytic MDSC, neutrophils, M1 and M2 macrophages) among CD45 + cells in both control and treated groups. The intratumoral immune cell infiltration was analyzed in five mice per condition. (I) scatter dot plots showing the percentage of tumor-infiltrating Treg (Foxp3 + ) CD4 + and CD8 + cells among total CD4 + and CD8 + populations in the different treatment groups. The activation status of DC (J) and pDC (K) was determined by flow cytometry. the expression of several surface markers (CD80, CD86, I-A/I-E, ILT3 and ICOSL) was assessed. Data represent the mean fluorescent intensity (MFI)±SEM of five independent experiments in each group (each individual data point is shown). (L) The apoptotic cancer cells (cleaved caspase 3 + ) were detected by immunohistochemistry and quantified using QuPath software. The number of positive cells was reported to tumor area (mm 2 ). The scale bar represents 100 µm. Asterisks indicate statistically significant differences (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). P values were determined using one-way ANOVA, followed by Bonferroni post-test (A, L), Fisher’s exact test (C, D) and one-way ANOVA followed by Dunnett’s multiple comparison post-test (F, G, H, I, J, K). ANOVA, analysis of variance; DC, dendritic cell; EP, ethyl pyruvate; HMGB1, high-mobility group box 1; i.p, intraperitoneal; METABRIC, Molecular Taxonomy of Breast Cancer International Consortium; pDC, plasmacytoid DC; RAP, RAGE antagonist peptide.

Article Snippet: Cell culture supernatant was then harvested and HMGB1 release by both human and mouse breast cancer cells was quantified by ELISA using the following commercially available kit: HMGB1 Detection kit (Chondrex).

Techniques: Staining, Flow Cytometry, Injection, Activation Assay, Expressing, Immunohistochemistry, Software

Journal: Journal for Immunotherapy of Cancer

Article Title: Extracellular HMGB1 blockade inhibits tumor growth through profoundly remodeling immune microenvironment and enhances checkpoint inhibitor-based immunotherapy

doi: 10.1136/jitc-2020-001966

Figure Lengend Snippet: Extracellular HMGB1 blockade enhances anti-PD-1-induced inhibition of tumor growth in vivo. (A) PD-1 mRNA expression ( PDCD1 gene) in the four major molecular subtypes of breast cancer was determined using the METABRIC public dataset. (B) Representative example of breast cancer stained for PD-1. Positive cells were observed in the epithelial component of the tumor as well as in the stroma surrounding cancer cells. (C) PD-1 + cell infiltration within tumor microenvironment was determined by computerized counting. Each point represents the number of positive cells/mm 2 for one independent tumor specimen. (D) Mouse breast cancer cells (4T1 and 67NR) were orthotopically injected into the mammary fat pad of immunocompetent BALB/c mice. Anti-PD-1 antibody was tested alone (i.p. injection of 200 µg at days 4, 7 and 11) and in combination with HMGB1 inhibitors (RAP (10 µM/kg) and EP (1 mM/kg), treatment at 3 day intervals). In parallel, the anticancer efficacy of these combination regimens was also compared with that displayed by each individual HMGB1 inhibitor used in monotherapy. The mean tumor volumes±SEM are represented. (E) The apoptotic cancer cells (cleaved caspase 3 + ) were detected by immunohistochemistry and quantified using QuPath software. The number of positive cells was reported to tumor area (mm 2 ). (F) The total number of (CD45 + ) immune cells per milligram of tumor was determined in the different treatment groups. (G) Scatter dot plots illustrating the percentage of each individual immune cell population (DC, PDC, CD4 + and CD8 + T cells, monocytic and granulocytic MDSC, neutrophils, M1 and M2 macrophages) among CD45 + cells in both control and treated groups. Reduced densities of granulocytic MDSC as well as an increase of M1 macrophages were especially observed in case of combination therapy. The intratumoral immune cells were analyzed in five mice per condition. (H) Scatter dot plots showing the percentage of tumor-infiltrating Treg (Foxp3 + ) CD4 + and CD8 + cells among total CD4 + and CD8 + populations in the different treatment groups. the activation status of DC (I) and pDC (J) was determined by flow cytometry. the expression of several surface markers (CD80, CD86, I-A/I-E, ILT3 and ICOSL) was analyzed. Data represent the mean fluorescent intensity (MFI)±SEM of 5 independent experiments in each group (each individual data point is shown). The scale bar represents 100 µm. Asterisks indicate statistically significant differences (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). P values were determined using one-way ANOVA followed by Bonferroni post-test (A, C, E) or Dunnett’s multiple comparison post-test (D, F, G, H, I, J). ANOVA, analysis of variance; DC, dendritic cell; HMGB1, high-mobility group box 1; METABRIC, Molecular Taxonomy of Breast Cancer International Consortium; MDSC, myeloid-derived suppressor cells; pDC, plasmacytoid DC; RAP, RAGE antagonist peptide.

Article Snippet: Cell culture supernatant was then harvested and HMGB1 release by both human and mouse breast cancer cells was quantified by ELISA using the following commercially available kit: HMGB1 Detection kit (Chondrex).

Techniques: Inhibition, In Vivo, Expressing, Staining, Injection, Immunohistochemistry, Software, Activation Assay, Flow Cytometry, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Extracellular HMGB1 blockade inhibits tumor growth through profoundly remodeling immune microenvironment and enhances checkpoint inhibitor-based immunotherapy

doi: 10.1136/jitc-2020-001966

Figure Lengend Snippet: A significant fraction of HMGB1 contained in tumor-extruded fluids is in its oxidized form and displays RAGE-dependent tolerogenic properties. (A, B) The ROS accumulation in both breast (4T1, 67NR, EpRas) and lung (TC-1) cancer cells used in the present study was assessed by flow cytometry. N-acetylcysteine (5 mM) and tert-butyl hydroperoxide (100 µM) were used as negative and positive controls, respectively. Results represent the means±SEM of four independent experiments (each individual data point is shown). (C) The redox state of extracellular HMGB1 contained in tumor-extruded fluids was analyzed by Western blot. All samples were directly alkylated in order to ‘freeze’ the redox state of HMGB1 molecules. Recombinant HMGB1 (0.5 µg) incubated with either H 2 O 2 or DTT (and then alkylated) were used as controls. (D) Oxidized/reduced-disulfide HMGB1 ratio (%) was calculated from the Western blot bands using ImageJ software. (E) DCs were incubated with terminally oxidized, fully reduced or disulfide HMGB1 for 24 hours before being stimulated with LPS for 24 hours. The expression of cell-surface molecules (CD80, CD83, CD86, HLA-DR, HLA-ABC and CCR7) was then measured by flow cytometry. All data were normalized to LPS-stimulated DC. Data represent the relative mean fluorescent intensity (MFI)±SEM of at least five independent experiments (each individual data point is shown). (F) DCs were incubated with terminally oxidized HMGB1 for 24 hours before being stimulated with LPS for 24 hours. When indicated, an inhibitor of RAGE (10 µM RAP) or TLR4 (2 µM LPS-RS) was added in the cell culture. The expression of DC activation markers was determined by flow cytometry. All data were normalized to LPS-stimulated DC. The relative MFI±SEM of 7 independent experiments are shown. Asterisks indicate statistically significant differences (*p<0.05, **p<0.01, ***p<0.001). P values were determined using one-way ANOVA, followed by Dunnett’s multiple comparison post-test (A, B, E, F). ANOVA, analysis of variance; DC, dendritic cell; HMGB1, high-mobility group box 1.

Article Snippet: Cell culture supernatant was then harvested and HMGB1 release by both human and mouse breast cancer cells was quantified by ELISA using the following commercially available kit: HMGB1 Detection kit (Chondrex).

Techniques: Flow Cytometry, Western Blot, Recombinant, Incubation, Software, Expressing, Cell Culture, Activation Assay

Journal: iScience

Article Title: Merkel cell polyomavirus T-antigens regulate DICER1 mRNA stability and translation through HSC70

doi: 10.1016/j.isci.2021.103264

Figure Lengend Snippet:

Article Snippet: Protein complexes were isolated with 40 μL of Dynabeads M-280 Streptavidin (Thermo Fisher Scientific, #11205D).

Techniques: Virus, Recombinant, Transfection, Electroporation, Bicinchoninic Acid Protein Assay, Reverse Transcription, Gene Expression, Polymerase Chain Reaction, Luciferase, Mutagenesis, In Vitro, Construct, shRNA, Software

Journal: Cell Reports

Article Title: Integrin-Mediated Macrophage Adhesion Promotes Lymphovascular Dissemination in Breast Cancer

doi: 10.1016/j.celrep.2019.04.076

Figure Lengend Snippet:

Article Snippet: 76kDa dextran fluorescein , Santa Cruz , #sc-263323.

Techniques: Recombinant, Liposomes, Enzyme-linked Immunosorbent Assay, Gene Expression, Derivative Assay, Construct, Software, shRNA

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

Journal: The Journal of Experimental Medicine

Article Title: The human CIB1–EVER1–EVER2 complex governs keratinocyte-intrinsic immunity to β-papillomaviruses

doi: 10.1084/jem.20170308

Figure Lengend Snippet: Identification of homozygous mutations affecting human CIB1 in a cohort of 24 EV patients. (A) Pedigrees of six kindreds affected by EV. Familial segregation of homozygous CIB1 mutations (m/m) in six consanguineous families indicating an AR pattern of inheritance with complete clinical penetrance. (B) Graphical representation of the CIB1 cDNA exon (c.CIB1) and protein (p.CIB1) structure with presentation of the EF-hand domains. The arrows at the top indicate the location of the cDNA positions affected by the CIB1 mutations found in the families, whereas those at the bottom indicate their consequences at protein level. *, stop codon; del, deletion; ins, insertion; fs, frameshift.

Article Snippet: RT-qPCR was then performed with TaqMan assays using exon-spanning probes for CIB1 (Hs01089679_m1), EVER1/TMC6 (Hs00273164_m1), or EVER2/TMC8 (Hs00380060_m1; Thermo Fisher Scientific) with normalization against GAPDH (4310884E), 18S (Hs99999901_s1), or RNaseP (4403326) as housekeeping genes by the ΔCT method.

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: The human CIB1–EVER1–EVER2 complex governs keratinocyte-intrinsic immunity to β-papillomaviruses

doi: 10.1084/jem.20170308

Figure Lengend Snippet: CIB1 levels in patient-derived cells and healthy skin biopsy specimens. (A) CIB1 mRNA levels were assessed by RT-qPCR in patient-derived LCLs for kindred A and P12 and in whole blood from P13. Each symbol represents the mean of three independent measurements per cell line/donor. Statistical significance (***, P < 0.001) was assessed in a one-way ANOVA followed by Dunnett’s multiple comparison test for kindred A. Levels were 47% lower than the corresponding control mean in P12 and 80% lower in P13. (B) CIB1 protein was detected in LCLs and PBMCs with a polyclonal antibody directed against the N terminus of CIB1. GAPDH and Akt served as loading controls. Cellular material from kindreds A and B was analyzed three times. PBMCs from kindred E were analyzed once. (C) CIB1 protein level in skin biopsy specimens from healthy donors were measured by IHC with a monoclonal antibody directed against CIB1. An isotype Ig was included as a control. These results are representative of two experiments, each performed on two patients. *, epidermis; >, hair shaft. Bars: (top) 100 μm; (middle and bottom) 50 μm. (D) CIB1 protein was detected in primary keratinocytes from three healthy controls (ctrl 1–3), kindreds A, C, and D, or in whole-skin lysates from kindred F. L, lesion. These results are representative of three and one independent experiments, respectively. Asterisk indicates a nonspecific band.

Article Snippet: RT-qPCR was then performed with TaqMan assays using exon-spanning probes for CIB1 (Hs01089679_m1), EVER1/TMC6 (Hs00273164_m1), or EVER2/TMC8 (Hs00380060_m1; Thermo Fisher Scientific) with normalization against GAPDH (4310884E), 18S (Hs99999901_s1), or RNaseP (4403326) as housekeeping genes by the ΔCT method.

Techniques: Derivative Assay, Quantitative RT-PCR, Comparison, Control

Journal: The Journal of Experimental Medicine

Article Title: The human CIB1–EVER1–EVER2 complex governs keratinocyte-intrinsic immunity to β-papillomaviruses

doi: 10.1084/jem.20170308

Figure Lengend Snippet: CIB1 forms a complex with EVER1 and EVER2. (A) CIB1 protein levels in LCLs derived from patients with loss-of-function mutations of EVER1 (D576*) and EVER2 (D362* or T150Mfs*3), and comparison with those in heterozygous carriers, positive controls (ctrl 1 and 2), P4 and P11, and patients with RHOH and MST1 deficiencies. (B) CIB1, EVER1, and EVER2 mRNA levels were assessed by RT-qPCR in controls ( n = 4), CIB1 m/m (P1–P6 and P12), EVER1 m/m, and EVER2 m/m ( n = 3 each) LCLs. Each symbol represents the mean of one cell line measured in three independent experiments. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparison tests relative to healthy controls. (C) CIB1 protein levels in LCLs derived from patients with loss-of-function mutations of EVER1 (D576*), EVER2 (T150Mfs*3), and a healthy control after reconstitution with WT EVER1 or EVER2 by retroviral transduction and stable selection. Asterisk indicates a nonspecific band. (D) CIB1, EVER1, and EVER2 mRNA levels were measured by RT-qPCR in LCLs derived from patients with loss-of-function mutations of EVER1 (D576*), EVER2 (T150Mfs*3), or CIB1 and a healthy control after reconstitution with WT EVER1 or EVER2 by retroviral transduction and stable selection. The data were first normalized against RNaseP as a housekeeping gene and then against an appropriate untransduced parental cell line by the ΔΔCt method. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparison test relative to the corresponding untransduced control. (B and D) ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (E) HEK293T cells were transfected with plasmids encoding CIB1-HA, FLAG-EVER1, and FLAG-EVER2 either separately or together; 24 h after transfection, samples were subjected to immunoprecipitation with FLAG (Fl)- or HA-specific antibodies (+). Samples incubated with nonspecific IgG served as specificity controls. Western blots were performed to detect coimmunoprecipitated CIB1-HA and FLAG-EVER1 or FLAG-EVER2. The immunoprecipitation of EVER1/2 and CIB1 was confirmed by reincubation with antibodies specific for FLAG and HA, respectively. The presence of all proteins was checked by Western blotting of an input sample taken before immunoprecipitation. GAPDH served as a loading control. vec, vector. (F) Healthy control keratinocytes were transfected with plasmids encoding CIB1-HA, FLAG-EVER1, and FLAG-EVER2 either alone or in combination; 24 h after transfection, cells were subjected to immunofluorescence imaging with Alexa Fluor 568–HA and Alexa Fluor 488–FLAG antibody combinations. DAPI was used for counterstaining. Colocalization was assessed by calculating Pearson’s correlation coefficient with Imaris software. Bar, 13 µm. The results shown are representative of three independent experiments.

Article Snippet: RT-qPCR was then performed with TaqMan assays using exon-spanning probes for CIB1 (Hs01089679_m1), EVER1/TMC6 (Hs00273164_m1), or EVER2/TMC8 (Hs00380060_m1; Thermo Fisher Scientific) with normalization against GAPDH (4310884E), 18S (Hs99999901_s1), or RNaseP (4403326) as housekeeping genes by the ΔCT method.

Techniques: Derivative Assay, Comparison, Quantitative RT-PCR, Control, Retroviral, Transduction, Selection, Transfection, Immunoprecipitation, Incubation, Western Blot, Plasmid Preparation, Immunofluorescence, Imaging, Software

Journal: The Journal of Experimental Medicine

Article Title: The human CIB1–EVER1–EVER2 complex governs keratinocyte-intrinsic immunity to β-papillomaviruses

doi: 10.1084/jem.20170308

Figure Lengend Snippet: Analysis of zinc signaling/levels and NF- k B activation in the presence and absence of CIB1. (A) HEK293T cells were transfected with plasmids encoding CIB1, EVER1, and EVER2 either alone or in combination and with a 4×MRE-dependent EGFP reporter construct. After 24 h, cells were stimulated overnight with PMA/ionomycin (10 ng/ml and 50 ng/ml, respectively) or zinc sulfate (ZnSO 4 ; 100 µM). The next day, cells were stained with 1 µg/ml DAPI to exclude dead cells, and GFP fluorescence was determined with an LSRII flow cytometer. The RRR with the value for vector-transfected cells was set at 100%. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparison test relative to the appropriate vector-transfected control (ns, P > 0.05; *, P < 0.05; ***, P < 0.001; n = 3). (B) Flow cytometric quantification of absolute amounts of labile zinc in LCLs derived from healthy controls, EVER1-, EVER2-, or CIB1-deficient patients, or in keratinocytes from P14 with 1 µM FluoZin-3 as described by . Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparison test relative to the healthy controls ( n = 3). (C) Kinetics of zinc flux in LCLs derived from healthy controls and EVER1-, EVER2-, or CIB1-deficient patients. Cells were loaded with 1 µM FluoZin-3 for 30 min. Fluorimetric measurement were performed on a Victor microplate reader. Baseline fluorescence was recorded every minute for 10 min. Cells were then loaded with 100 µM ZnSO 4 and recorded for 15 min. The specificity of the zinc signal was confirmed by adding the calcium-specific chelator BAPTA before the quenching of the signal with the zinc-specific chelator TPEN and recording for 20 min. No significant effect of genotype was detected in two-way repeat-measures ANOVA ( n = 3). (D) HEK293T cells were transfected with plasmids encoding CIB1-FLAG, FLAG-EVER1, and EVER2 either alone or in combination. Cells were stimulated with 50 ng/ml TNFα 6 h after transfection, incubated overnight, and then harvested and processed for Western blotting. Membranes were probed for the canonical NF-κB component (p)IκBa and the noncanonical NF-κB component p100/p52. Expression of the constructs used for transfection was verified by incubation with a FLAG-specific antibody. GAPDH served as a loading control ( n = 3). (E) Primary keratinocytes from unrelated donors (controls 1 and 2), a healthy family member from kindred A1 carrying the mutation in a heterozygous state (A1.viii.2), and one patient each from kindreds A1 and C were stimulated with 10 ng/ml TNFα for 5, 10, or 20 min and then harvested and processed for Western blotting. Membranes were probed for the canonical NF-κB component (p)IκBa. CIB1 levels were assessed with a polyclonal antibody. GAPDH served as a loading control. vec, empty vector ( n = 3).

Article Snippet: RT-qPCR was then performed with TaqMan assays using exon-spanning probes for CIB1 (Hs01089679_m1), EVER1/TMC6 (Hs00273164_m1), or EVER2/TMC8 (Hs00380060_m1; Thermo Fisher Scientific) with normalization against GAPDH (4310884E), 18S (Hs99999901_s1), or RNaseP (4403326) as housekeeping genes by the ΔCT method.

Techniques: Activation Assay, Transfection, Construct, Staining, Fluorescence, Flow Cytometry, Plasmid Preparation, Comparison, Control, Derivative Assay, Incubation, Western Blot, Expressing, Mutagenesis

Journal: The Journal of Experimental Medicine

Article Title: The human CIB1–EVER1–EVER2 complex governs keratinocyte-intrinsic immunity to β-papillomaviruses

doi: 10.1084/jem.20170308

Figure Lengend Snippet: Focal adhesion formation and scratch wounding in CIB1-deficient keratinocytes. (A) Primary keratinocytes from two donors, a healthy member of kindred A (A1.viii.2; CIB1 wt/m), and P5 were allowed to adhere to fibronectin-coated plates (10 µg/ml) for the times indicated and were then harvested and processed for Western blotting. The membranes were probed for the focal adhesion (FA) components vinculin and (p)FAK. GAPDH served as a loading control. (B and C) Primary keratinocytes from two donors, a healthy member of kindred A (A1.viii.2), and P5 were allowed to adhere to fibronectin-coated coverslips (10 µg/ml) for 24 h. Cells were fixed and stained with vinculin- and pFAK-specific antibodies. Z stacks were acquired with a confocal microscope. (B) Representative image for automated surface detection for control 1. Bar, 40 µm. (C) The volume (size), fluorescence intensity, and morphology of the vinculin- and pFAK-positive structures were automatically determined with Imaris. Values were normalized against the total number of surfaces detected. Statistical significance was assessed in one-way ANOVA followed by Dunnett’s multiple comparison test relative to control 1. No significant differences were detected for any of the parameters tested (P > 0.05). (D–F) Primary keratinocytes from two donors, a healthy member of kindred A (A1.viii.2; CIB1 wt/m), and P5 were allowed to adhere to fibronectin-coated plates (1 µg/ml) overnight. Scratch wounds were created with a pipette tip, and wound closure was monitored by live-cell microscopy every 20 min for 16 h. Bar, 283 µm. Wound closure was then quantified automatically with the MiToBo plugin in ImageJ with measurement of the cell-free area normalized against the starting point (set to 100%; D and E). Statistical significance was assessed by two-way repeated-measures ANOVA. Differences between controls 1 and 2, CIB1 wt/m, and P5 were statistically significant. Differences between control 2, CIB1 wt/m, and P5 were not significant. (F) For confirmation of the accuracy of the results obtained with the plugin, the total cell-free area at the start and end points was determined manually. The data shown are the means of two (controls 1 and 2) or three (CIB1 wt/m and P5) independent experiments with at least six data points acquired per set. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparison test relative to controls 1 or 2. (ns, P > 0.05; ***, P < 0.001).

Article Snippet: RT-qPCR was then performed with TaqMan assays using exon-spanning probes for CIB1 (Hs01089679_m1), EVER1/TMC6 (Hs00273164_m1), or EVER2/TMC8 (Hs00380060_m1; Thermo Fisher Scientific) with normalization against GAPDH (4310884E), 18S (Hs99999901_s1), or RNaseP (4403326) as housekeeping genes by the ΔCT method.

Techniques: Western Blot, Control, Staining, Microscopy, Fluorescence, Comparison, Transferring

Journal: The Journal of Experimental Medicine

Article Title: The human CIB1–EVER1–EVER2 complex governs keratinocyte-intrinsic immunity to β-papillomaviruses

doi: 10.1084/jem.20170308

Figure Lengend Snippet: PLA and coimmunoprecipitation in HaCaT. (A) HaCaT cells were transfected with plasmids encoding FLAG-HPV5 E1, E2, E6, and E7, FLAG-HPV16 E1, E2, E5, E6, and E7, FLAG-HPV4 E8, FLAG–CRPV E8, and CIB1-HA alone or in combination. The day after transfection, samples were plated on microscopy slides, allowed to adhere, fixed in acetone, permeabilized, and subjected to Duolink PLAs with rabbit-HA– and mouse-FLAG–specific antibodies. Z stacks were acquired with a widefield microscope, and PLA-positive sites (defined as structures >0.35 µM 2 ) were scored with Imaris software for 15–50 cells per condition. These pooled results were obtained in two independent experiments. The orange bars indicate the mean. (B) HaCaT cells were transfected with plasmids encoding CIB1-HA and the FLAG-tagged HPV E ORFs scoring positive in the PLA in A. 1 d after transfection, samples were subjected to immunoprecipitation (IP) with FLAG-specific antibodies. Western blots were performed to detect coimmunoprecipitated HPV5 E1, HPV16 E2, E5, HPV4 E8, and CRPV E8. The immunoprecipitation of CIB1 was confirmed by reincubation with a FLAG-specific antibody. The presence of all proteins was checked by Western blotting analysis on an input sample taken before immunoprecipitation. GAPDH served as a loading control ( n = 3). vec, vector.

Article Snippet: RT-qPCR was then performed with TaqMan assays using exon-spanning probes for CIB1 (Hs01089679_m1), EVER1/TMC6 (Hs00273164_m1), or EVER2/TMC8 (Hs00380060_m1; Thermo Fisher Scientific) with normalization against GAPDH (4310884E), 18S (Hs99999901_s1), or RNaseP (4403326) as housekeeping genes by the ΔCT method.

Techniques: Transfection, Microscopy, Software, Immunoprecipitation, Western Blot, Control, Plasmid Preparation

Journal: The Journal of Experimental Medicine

Article Title: The human CIB1–EVER1–EVER2 complex governs keratinocyte-intrinsic immunity to β-papillomaviruses

doi: 10.1084/jem.20170308

Figure Lengend Snippet: Mechanism underlying EV. (A) In the general population, the CIB1–EVER1–EVER2 complex restricts the transcription of minichromosome of β-HPV, leading to the absence of clinical manifestation. The proteins E5 and E8 expressed by the other cutaneous HPVs (α2-, α4-, γ-, and μ-HPVs) are able to antagonize the CIB1–EVER1–EVER2 complex. However, additional restriction factors are probably contributing to the absence of HPV lesions in the vast majority of people. (B) In EV patients, the lack of CIB1–EVER1–EVER2 permits the transcription of β-HPV minichromosome, which leads to the development of EV lesions on the skin. However, the probable presence of additional restriction factors against cutaneous HPVs other than β-HPVs accounts for their normal control, which does not differ from the general population.

Article Snippet: RT-qPCR was then performed with TaqMan assays using exon-spanning probes for CIB1 (Hs01089679_m1), EVER1/TMC6 (Hs00273164_m1), or EVER2/TMC8 (Hs00380060_m1; Thermo Fisher Scientific) with normalization against GAPDH (4310884E), 18S (Hs99999901_s1), or RNaseP (4403326) as housekeeping genes by the ΔCT method.

Techniques: Control