Journal: Frontiers in Immunology

Article Title: AHR Regulates NK Cell Migration via ASB2–Mediated Ubiquitination of Filamin A

doi: 10.3389/fimmu.2021.624284

Figure Lengend Snippet: Aryl hydrocarbon receptor (AHR) deficient natural killer (NK) cells have low migratory capacity. (A) Tumor infiltration by mouse NK cells. Splenic NK cells from Ahr +/+ and Ahr -/- mice were tail vein injected (5x10 6 /mice) into MOC2-bearing NSG mice (n=3). After 24 h, tumors were dissociated, and the amount of NK cells was determined by FACS. Graph is expressed as mean ± SEM. *p < 0.05. (B, C) NK cell migration is decreased in AHR-deficient NK cells. Purified NK cells from Ahr +/+ and Ahr -/- mice were cultured with IL-2 (1,000 U/mL) for 7 days, then analyzed by time-lapse microscopy. Images were acquired every 5 min for 4 h. (B) Average migration speed is presented, 50 cells per group. *p < 0.05. (C) Migration distance of Ahr +/+ and Ahr -/- NK cells was analyzed with time-lapse microscopy (n=10). (D) The invasion ability of Ahr +/+ and Ahr -/- NK cells was measured at 24 h after seeding them on the Matrigel chamber (n=10). Results are expressed as the number of invading cells per 0.16 mm 2 . All experiments were repeated at least three times, and the result is expressed as mean ± SEM. *p < 0.05. (E–G) NK-92MI cells expressing control shRNA or AHR shRNA were seeded on 5 μg/mL fibronectin. Cells were then imaged in brightfield by live confocal microscopy 2 tp/min and manually tracked. Graphs show the results from one experiment, of three independent experiments, showing length (E) , displacement length (F), and velocity of cells (G) (n=18–20, ****p < 0.0001 by Mann-Whitney test). (H) NK-92MI cells were treated with AHR antagonist CH223191 (1uM) or control condition (DMSO) for 2 days, then homotypic aggregation was analyzed using in an optical microscope. One representative example of three different assessments is shown.

Article Snippet: For some experiments, to assess the involvement of AhR, media was supplemented with either DMSO (ATCC; Cat# 4-X), 200 nM FICZ (ENZO; Cat# BML-GR206-0100) or 3 μM CH-223191 (Sigma-Aldrich; Cat# C8124-5MG).

Techniques: Injection, Migration, Purification, Cell Culture, Time-lapse Microscopy, Expressing, Control, shRNA, Confocal Microscopy, MANN-WHITNEY, Microscopy

Journal: Frontiers in Immunology

Article Title: AHR Regulates NK Cell Migration via ASB2–Mediated Ubiquitination of Filamin A

doi: 10.3389/fimmu.2021.624284

Figure Lengend Snippet: Aryl hydrocarbon receptor (AHR) regulates expression of ubiquitin ligase subunit ASB2. (A) Splenic natural killer (NK) cells from Ahr +/+ and Ahr -/- mice were cultured in the presence of IL-2 (1,000 U/mL) for 8 days. Then, their gene expression was assessed by microarray. Heatmap shows microarray signal intensity of the top 40 differentially expressed genes (n=3 mice per group). Asterix (*) denotes genes with at least one AHR binding domain in their regulatory region. (B) Asb2 mRNA expression was assessed on mouse splenic NK cells cultured in the presence of IL-2 (1,000 U/mL) and FICZ (200 nM) or vehicle control (DMSO) for 7 days (n=3 mice per group). (C) Human NK-92MI cells were cultured with AHR agonist FICZ (200 nM), antagonist CH-223191 (1 uM), or vehicle control (DMSO) for 24 h, then Asb2 mRNA expression was determined. (B, C) Graphs are expressed as mean ± SEM. *p < 0.05. (D) Sorted human CD56dim and CD56bright NK cells were cultured with AHR agonist FICZ (200 nM) or vehicle control (DMSO) for 24 h, then ASB2 mRNA expression was determined. (E) Chromatin Immuno-Precipitation assay analyzing the binding of AHR to the promoter region of Asb2 gene. DNA fragments were immunoprecipitated using anti-AHR or anti-H3 antibodies (positive control), or protein A bead only as a negative control; and the immunoprecipitated DNA fragments were amplified using corresponding gene-specific promoter primers. Input: PCR performed using DNA before immunoprecipitation. ATG represents the location of start codon. Arrows labeled F and R represent the gene-specific primers and their positions on target gene promoter. All ChIP experiments were repeated at least three times. (F) AHR-dependent increase of Asb2 promoter activity. pBV-Luc-Asb2-1K (-973/-1 from ATG) vector was transfected into HEK-293 cells with mAhR or hAhR expression vectors, and the luciferase activity was measured after 48 h with a Luminometer. Experiments were performed three times. For statistical comparisons, paired two-tailed Student t-test was used. (G) FICZ increases Asb2 promoter activity. pBV-Luc-Asb2-1K vector and mouse AhR expression vector were transfected into HEK-293 cells in combination, and 1 day later, HEK-293 cells were treated with FICZ (200 nM) for 24 h. Cells were harvested and luciferase activity was measured with a Luminometer. pBV-Luc-Asb2-1K vector-transfected cells treated with DMSO or FICZ (200 nM) were used as control. Experiments were performed three times. For statistical comparisons, paired two-tailed Student t-test was used.

Article Snippet: For some experiments, to assess the involvement of AhR, media was supplemented with either DMSO (ATCC; Cat# 4-X), 200 nM FICZ (ENZO; Cat# BML-GR206-0100) or 3 μM CH-223191 (Sigma-Aldrich; Cat# C8124-5MG).

Techniques: Expressing, Ubiquitin Proteomics, Cell Culture, Gene Expression, Microarray, Binding Assay, Control, Chromatin Immunoprecipitation, Immunoprecipitation, Positive Control, Negative Control, Amplification, Labeling, Activity Assay, Plasmid Preparation, Transfection, Luciferase, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: AHR Regulates NK Cell Migration via ASB2–Mediated Ubiquitination of Filamin A

doi: 10.3389/fimmu.2021.624284

Figure Lengend Snippet: ASB2 is involved in the aryl hydrocarbon receptor (AHR)-mediated regulation of natural killer (NK) cell migration. (A) The phenotype of ASB2 knocked-down or control NK-92MI cells was analyzed using in an optical microscope. One representative example of three different assessments is shown. (B) Migration ability of NK cells was analyzed using time-lapse video microscopy. Representative graph shows migration distance of 50 randomly selected NK-92MI cells expressing ASB2 shRNA or control shRNA, in the presence of CH-223191 (1 μM) or vehicle control (DMSO), on a 10% Matrigel chamber. The quantitation of migration distance was done for 6 h, with 5 min intervals. One representative example of three different assessments is shown. (C) Cell invasion was analyzed using ASB2 knocked-down or control NK-92MI cells. The invasion capacity was measured on day 3 and 4 after seeding. (top) Images show results from a representative experiment; (bottom) graph shows cumulative results from 3 independent experiments expressed as mean ± SD. (D) Tumor infiltration by NK-92MI cells. NK-92MI-GFP cells expressing ASB2 shRNA or control shRNA were tail vein injected (3x10 6 /mice) into UM-SCC-103-bearing NSG mice (n=3). After 24 h, tumors were dissociated, and the amount of infiltrating NK cells was determined by FACS. Graph shows the amount of infiltrating NK cells per 3x10 5 dissociated cells and is expressed as means ± SEM. * p < 0.05.

Article Snippet: For some experiments, to assess the involvement of AhR, media was supplemented with either DMSO (ATCC; Cat# 4-X), 200 nM FICZ (ENZO; Cat# BML-GR206-0100) or 3 μM CH-223191 (Sigma-Aldrich; Cat# C8124-5MG).

Techniques: Migration, Control, Microscopy, Expressing, shRNA, Quantitation Assay, Injection

Journal: Frontiers in Immunology

Article Title: AHR Regulates NK Cell Migration via ASB2–Mediated Ubiquitination of Filamin A

doi: 10.3389/fimmu.2021.624284

Figure Lengend Snippet: ASB2 regulates filamin A protein level in natural killer (NK) cells. (A) NK-92MI cells were cultured in the presence of CH-223191 (1 μM) or vehicle control (DMSO) for 2 days, then the amount of FLNA , FLNB , and FLNC mRNA was determined by qRT-PCR. Graphs show mRNA expression, measured in triplicates, and are shown as mean ± SEM. (B) The invasion ability of NK-92MI cells stable transfected with FLNA shRNA or control shRNA, was measured at 24 h after seeding on the Matrigel chamber. Results are expressed as the number of invading cells per 0.16 mm 2 . All experiments were repeated at least three times, and the result is expressed as mean ± SEM. p value is shown. (C) NK cells from Ahr +/- and Ahr -/- mice were cultured with IL-2 (1,000 U/ml) for 7 days and then cultured in the presence of FICZ (200 nM), CH-223191 (1 μM), or vehicle control (DMSO) for two additional days. FLNA level was analyzed by western blotting. (D) B6 mice were injected intraperitoneally with FICZ (50 μM/mice), CH-223191 (500 μM/mice) or vehicle control (DMSO). After 2 days, the spleens were collected and CD8 - NKp46 + NK cells were FACS sorted and cultured with IL-2 (1,000 U/ml) for 2 days. Then cells were assessed for FLNA level by western blotting. (E) The level of FLNA was analyzed on NK-92MI stable transfected with ASB2 shRNA, ASB2 over-expression, or control vectors by western blotting. (F) ASB2 protein expression was confirmed in NK92MI cells transfected to overexpress ASB2 . (G) Ubiquitination analysis. NK-92MI cells were cultured in the presence of FICZ (200 nM). At different time-points during the culture, NK-92MI were collected, lysed, immunoprecipitated with anti-FLNA antibody and blotted with anti-ubiquitin antibody . (A–F) Results from one representative experiment are shown, from at least three different experiments.

Article Snippet: For some experiments, to assess the involvement of AhR, media was supplemented with either DMSO (ATCC; Cat# 4-X), 200 nM FICZ (ENZO; Cat# BML-GR206-0100) or 3 μM CH-223191 (Sigma-Aldrich; Cat# C8124-5MG).

Techniques: Cell Culture, Control, Quantitative RT-PCR, Expressing, Transfection, shRNA, Western Blot, Injection, Over Expression, Ubiquitin Proteomics, Immunoprecipitation

Journal: Nature Communications

Article Title: Hypoxia induced responses are reflected in the stromal proteome of breast cancer

doi: 10.1038/s41467-023-39287-7

Figure Lengend Snippet: Multivariate survival analysis (proportional hazards regression model) of breast cancer patients (METABRIC-Discovery cohort)

Article Snippet: Of relevance for secretome studies, we found that 40 of the PAM50 signature genes/proteins have been reported in serum or plasma (plasma proteome database; PPD: http://www.plasmaproteomedatabase.org/ ) , and/or the Human Protein Atlas—blood protein (Human Protein Atlas proteinatlas.org) .

Techniques:

Journal: International Journal of Biological Sciences

Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

doi: 10.7150/ijbs.29562

Figure Lengend Snippet: The expression of SMARCAD1 is significantly higher in pancreatic cancer and positively correlated with poor prognosis. A. The mRNA expression level of SMARCAD1 in pancreatic cancer is much higher than that of normal tissues from GSE16515, GSE11838 and GSE15471. B-C. Expression levels of SMARCAD1 by immunohistochemistry performed with tissue microarray of PC (n=69) and adjacent normal tissues (n=68). Representative images showed positive expression of SMARCAD1 in PC and negative expression in paired normal tissues, respectively. Scale bars=50μm. D. Kaplan-Meier analysis shows the correlation between SMARCAD1 expression and overall survival in patients. Patients with high SMARCAD1 expression had poorer overall survival than those with low expression. *p<.05, **p<.01.

Article Snippet: The specimens were incubated with anti-SMARCAD1 antibody (1:50), and staining results were observed with a Nikon ECLIPSETs2R microscope.

Techniques: Expressing, Immunohistochemistry, Microarray

Journal: International Journal of Biological Sciences

Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

doi: 10.7150/ijbs.29562

Figure Lengend Snippet: SMARCAD1 enhances proliferation of PANC-1 cells. A-B. The efficiency of SMARCAD1 knockdown (A) or overexpression (B) in PANC-1 cells was detected by western blotting. β-actin was used as an internal control. C-D. CCK8 assay was performed to determine the proliferation of PANC-1 cells with SMARCAD1 knockdown (C) or overexpression (D) at the indicated time points after plated. Cell viability was measured at 450nm. E-F. The effect of SMARCAD1 knockdown (E) or overexpression (F) on Colony-forming of PANC-1 cells was shown in the top panels. Number of foci was counted as shown in the bottom panels. All data were presented as mean ±SEM. *p<.05, **p<.01.

Article Snippet: The specimens were incubated with anti-SMARCAD1 antibody (1:50), and staining results were observed with a Nikon ECLIPSETs2R microscope.

Techniques: Knockdown, Over Expression, Western Blot, Control, CCK-8 Assay

Journal: International Journal of Biological Sciences

Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

doi: 10.7150/ijbs.29562

Figure Lengend Snippet: SMARCAD1 promotes migration and invasion of PANC-1 cells. A-B. Effect of SMARCAD1 knockdown (A) or SMARCAD1 overexpression (B) on cell migration was detected by wound healing at indicated time points after scratching. The wound healing was measured by ImageJ software. C-D. Motility ability of PANC-1 cells with SMARCAD1 depletion (C) or overexpression (D) was assessed by transwell assay at 24h. Representative images of migration were photographed at 24h (Top panel). The number of migrated cells was counted from 5 randomly selected fields under microscope (Bottom panel). E-F. Invasion ability of PANC-1 cells with SMARCAD1 depletion (E) or overexpression (F) was assessed by transwell assay at 48h. Representative images of invasion were photographed at 48h (Top panel). The number of invaded cells was counted from 5 randomly selected fields under microscope (Bottom panel). Scale bars=150um. Data were presented as mean ±SEM. *p<.05, **p<.01.

Article Snippet: The specimens were incubated with anti-SMARCAD1 antibody (1:50), and staining results were observed with a Nikon ECLIPSETs2R microscope.

Techniques: Migration, Knockdown, Over Expression, Software, Transwell Assay, Microscopy

Journal: International Journal of Biological Sciences

Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

doi: 10.7150/ijbs.29562

Figure Lengend Snippet: SMARCAD1 induces EMT in PANC-1 cells. A-B. The morphology changes of PANC-1 cells: cells lose contact with each other with SMARCAD1 depletion (A) or gain more contact with SMARCAD1 overexpression (B), Scale bars=250μm. C-D. Changes in mRNA level of EMT relative markers were tested by Quantitative real-time PCR in SMARCAD1 knockdown (C) or overexpression (D) cells. The results were presented as mean ±SEM. All values were normalized to the level (=1) in NC or control cells. *p<.05, **p<.01. (D). E-F. The protein levels of EMT relative markers in SMARCAD1 knockdown (E) or overexpression (F) cells were assessed by western blotting. β-actin was used as an internal control.

Article Snippet: The specimens were incubated with anti-SMARCAD1 antibody (1:50), and staining results were observed with a Nikon ECLIPSETs2R microscope.

Techniques: Over Expression, Real-time Polymerase Chain Reaction, Knockdown, Control, Western Blot

Journal: International Journal of Biological Sciences

Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

doi: 10.7150/ijbs.29562

Figure Lengend Snippet: SMARCAD1-induced EMT was regulated by Wnt/beta-catenin signaling pathway. A-B. The mRNA level of β-catenin was detected by Quantitative real-time PCR in PANC-1 cells with SMARCAD1 knockdown (A) or overexpression (B) respectively. The data were presented as mean ±SEM. All values were normalized to the level (=1) in NC or control cells. *p<.05, **p<.01. C-D. β-catenin, cyclin-D1, c-Myc and survivin protein levels were assayed by western blotting in PANC-1 cells with SMARCAD1 knockdown (C) or overexpression (D) respectively. E. PANC-1 cells with SMARCAD1 depletion were treated with CHIR99021 (6μM/ml) for 24h. The protein levels of EMT markers and Wnt/β-catenin target genes (β-catenin, cyclin-D1, c-Myc and survivin) were detected by western blotting. β-actin was used as an internal control.

Article Snippet: The specimens were incubated with anti-SMARCAD1 antibody (1:50), and staining results were observed with a Nikon ECLIPSETs2R microscope.

Techniques: Real-time Polymerase Chain Reaction, Knockdown, Over Expression, Control, Western Blot

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: HNRNPM expression is increased in HCC and fetal liver tissues and is associated with prognosis. A , Normalized (Norm) HNRNPM expression levels during mouse liver development from GSE57824 data. B , HNRNPM expression levels during mouse liver development from GSE13149 data. C , Western blot analysis of HNRNPM protein levels in human fetal liver and adult liver tissues. D , Real-time qPCR analysis of HNRNPM mRNA levels in human fetal liver and adult liver tissues. Data are mean ± standard deviation of n = 3 independent samples. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001 by the Student t test. E , Norm HNRNPM expression in HCC and normal liver tissues. ∗∗ P < .01 by the Student t test. F , Real-time qPCR analysis of HNRNPM mRNA levels in 60 paired HCC and normal liver tissues. G , Representative images of HNRNPM by IHC in HCC and normal tissues. H , Kaplan-Meier analysis of HNRNPM in HCC cohort. I , Kaplan-Meier analysis of HNRNPM in TCGA cohort.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Expressing, Western Blot, Standard Deviation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: HNRNPM was associated with clinopathological characteristics and poor prognosis in patients with HCC. A , Oncomine analysis showed the prognostic splicing factors from TCGA datasets. B , The selected prognostic splicing factors validated by real-time PCR in portal vein tumor thrombosis (PVTT) HCC, non-PVTT HCC, and normal liver tissues. C , The HNRNPM protein expression in metastasis and metastasis-free HCC tissues. D , The HNRNPM protein expression in tumor grade I/II and III/IV. E , The HNRNPM protein expression in no-microvascular invasion and microvascular invasion HCC tissues. F , The relative HNRNPM expression in tumor stage I/II/III/IV. G , The correlation analysis between Ki-67 and HNRNPM in TCGA database. H , Kaplan-Meier analyses of the correlations between HNRNPM level and overall survival in HCC tumor stage I/II and III/IV from our HCC cohort. I , Kaplan-Meier analyses of the correlations between HNRNPM level and OS in HCC tumor grade I/II and III/IV from our HCC cohort.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Real-time Polymerase Chain Reaction, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: The Association of HNRNPM Expression With Clinical Characteristics in 240 Patients With HCC

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: The Association of HNRNPM Expression With Clinical Characteristics in 371 Patients With HCC

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Expressing, Virus

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: Univariate and Multivariate Cox Regression Analysis of Overall Survival for HNRNPM (n = 240)

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: Univariate and Multivariate Cox Regression Analysis of Overall and Disease-free Survival for HNRNPM (n = 370) From TCGA Database

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: Cell stem cell transcriptional factors SOX2 and OCT4 bind with promoter and upregulate the expression of HNRNPM. A , The basic expression of HNRNPM in different HCC cell lines. B-C , Western blot analysis of HNRNPM expression when overexpressing ( B ) or depletion of ( C ) OCT4 and SOX2. D , The predicted binding site for OCT4 and SOX2 with HNRNPM promoter. E , OCT4 directly binds with HNRNPM promoter by ChIP assays and luciferase assays. F , SOX2 directly binds with HNRNPM promoter by ChIP assays and luciferase assays. G-H , Correlation analysis between OCT4 ( G ), SOX2 ( H ), and HNRNPM from TCGA database.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Expressing, Western Blot, Binding Assay, Luciferase

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: The tumorigenesis effects of HNRNPM overexpression in MHCC97L and HepG2 cells. A-B , The mRNA and protein levels of HNRNPM in MHCC97L ( A ) and HepG2 cells ( B ) stably overexpressing HNRNPM. C-D , The cell proliferation by CCK-8 assays stably MHCC97L ( C ) and HepG2 cells ( D ) stably overexpressing HNRNPM. ∗∗∗∗ P < .0001 as compared with control. E-F , The cell apotosis by flow cytometry stably MHCC97L ( E ) and HepG2 cells ( F ) stably overexpressing HNRNPM. Data were from 3 independent experiments. ∗∗ P < .01 by the Student t test. G-H , The in vivo effects in BALB/c nude mice in MHCC97L ( G ; n = 6) and HepG2 cells ( H ; n = 6) stably overexpressing HNRNPM. ∗∗ P < .01 by the Student t test. I , The CSC frequency was determined from a limiting dilution assay performed with HCC cells depleting HNRNPM from the third transplant recipient mice (n = 6). The ELDA web tool was used to calculate the frequency of CSCs.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Over Expression, Stable Transfection, CCK-8 Assay, Control, Flow Cytometry, In Vivo, Limiting Dilution Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: HNRNPM drives HCC tumorigenesis and maintains CSC properties. A-B , Sphere formation and limiting dilution assays when overexpressed HNRNPM in MHCC97L and HepG2 cells. ∗ P < .05; ∗∗ P < .01 by the Student t test. The number of spheroids formed as a fraction of the number of cells seeded per well is given. Data are from 3 independent experiments. C-D , Cell cycle detected by flow cytometry when overexpressed HNRNPM in MHCC97L and HepG2 cells. ∗ P < .05; ∗∗ P < .01 by the Student t test. E-F , Colony formation assay when overexpressed HNRNPM in MHCC97L and HepG2 cells. ∗ P < .05; ∗∗ P < .01 by the Student t test. G-H , Cell migration assay when overexpressed HNRNPM in MHCC97L and HepG2 cells. ∗ P < .05 by the Student t test. I , Cell invasion assays when overexpressed HNRNPM in MHCC97L and HepG2 cells. Results are presented as mean ± standard error of the mean, n = 3. ∗ P < .05 by the Student t test.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Flow Cytometry, Colony Assay, Cell Migration Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: The oncofetal properties of HNRNPM in hepatocyte differentiation model. A , The model scheme in hepatocyte differentiation model. B , The expression of OCT4, E2F1, SOX2, and HNRNPM in different stages from hepatocyte differentiation model. C , The correlation analysis between HNRNPM and E2F1 from TCGA databases. D , The potential binding site for E2F1 to HNRNPM promoter. E , E2F1 directly bind with HNRNPM promoter by ChIP assays. Data were from 3 independent experiments. ∗∗ P < .01 by the Student t test.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Expressing, Binding Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: HNRNPM is required for tumorigenesis of HCC cells. A , The mRNA and protein levels of HNRNPM in MHCC97H cells stably depleting HNRNPM. B , The protein levels of HNRNPM by immunofluorence stably depleting HNRNPM. C , Sphere formation and limiting dilution assays when depleting HNRNPM in MHCC97H cells. The number of spheroids formed as a fraction of the number of cells seeded per well is given. Data are from 3 independent experiments. ∗∗ P < .01 by the Student t test. D , The cell proliferation by CCK-8 assays stably depleting HNRNPM in MHCC97H cells. Results are presented as mean ± standard error of the mean, n = 3. ∗ P < .05; ∗∗ P < .01 by the Student t test. E , The cell apotosis by flow cytometry stably depleting HNRNPM in MHCC97H cells. Results are presented as mean ± standard error of the mean, n = 3. ∗ P < .05; ∗∗ P < .01 by the Student t test. F , Cell cycle detected by flow cytometry when depleting HNRNPM in MHCC97H cells. G , Colony formation assay when depleting HNRNPM in MHCC97H cells. Results are presented as mean ± standard error of the mean, n = 3. ∗ P < .05; ∗∗ P < .01 by the Student t test. H , Cell migration assay when depleting HNRNPM in MHCC97H cells. Results are presented as mean ± standard error of the mean, n = 3. ∗∗∗ P < .001 by the Student t test. I , Cell invasion assays when depleting HNRNPM in MHCC97H cells. J-K , The in vivo effects in BALB/c nude mice (n = 6 per group) when overexpressed and depleted HNRNPM. Results are presented as mean ± standard error of the mean, n = 6. ∗ P < .05; ∗∗ P < .01 by the Student t test. L-M , The number of liver metastasis in BALB/c nude mice when overexpressed and depleted HNRNPM. Results are presented as mean ± standard error of the mean, n = 6. ∗ P < .05; ∗∗ P < .01 by the Student t test. N , The CSC frequency was determined from a limiting dilution assay performed with HCC cells from the third transplant recipient mice. The ELDA web tool was used to calculate the frequency of CSCs.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Stable Transfection, CCK-8 Assay, Flow Cytometry, Colony Assay, Cell Migration Assay, In Vivo, Limiting Dilution Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: The genome-wide landscape and global alternative splicing of HNRNPM. A , Kyoto Encyclopedia of Genes and Genomes analysis of HNRNPM-targeted splicing events. B , Quantification of the different AS events regulated by HNRNPM. A3SS , alternative 3′ splicing site; A5SS , alternative 5′ splicing site; MXE , mutually exclusive exon; RI , retained intron; SE , skipped exon. ∗∗∗ P < .001 by the Student t test. C-D , The quantification of significant AS events regulated by HNRNPM ( P < .05). E , HNRNPM-RIP-seq peaks were enriched in 5′UTR, promoter and 3′ UTR. All RIP-seq peaks were categorized according to the distribution on different genomic elements andcompared with the genomic background. F , De novo motif analysis identifying GU-repeat motif as the only enriched motif within the top HNRNPM RIP-seqpeaks. G , Schematic diagram of MBD2 molecular model. H , The RIP experiment showed HNRNPM directly binded with MBD2. I , The shift of MBD2a and MBD2c between HNRNPM overexpressed stably transduced and control MHCC97H cells. J , The shift of MBD2a and MBD2c between HNRNPM shRNA stably transduced and control MHCC97H cells. K , The RMMs of HNRNPM bind to MBD2 by RIP experiments. L , The potential binding of HNRNPM to MBD2 pre-mRNA by CLIP assay.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Genome Wide, Alternative Splicing, Stable Transfection, Control, shRNA, Binding Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: The Significant Alternative Splicing Events by Comparing Depletion of HNRNPM With Wild-type HCC Cells

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Alternative Splicing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: The Results of HNRNPM-RIP Analysis

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: The Intersection Results of HNRNPM-RIP Analysis and Transcriptomic Sequencing

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques:

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: DNA methylation controls MBD2-mediated FZD3 transcription. A , The shematic diagram of HNRNPM domains. B , The specific binding site for MBD2 with HNRNPM by CLIP assay. C , The luciferase assay for FZD3 transcription activity when overexpressing MBD2a or MBD2a and MBD2c. Data were from three independent experiments. ∗ P < .05. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test. D-H , qPCR analysis of MBD2a, MBD2c, FZD3, β-catenin, and Snail1 mRNA transcripts in MHCC97H cells stably expressing NC, shRNAs targeting HDAC1, HDAC2, RBBP7, or MTA2. Immunoblot analysis showed the knockdown efficiency of shRNAs targeting HDAC1, HDAC2, RBBP7, or MTA2 in MHCC97H cells. Data were from 3 independent experiments. ∗ P < .05. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test. I-J , β-catenin promotes the expression of OCT4 ( I ) and SOX2 ( J ) by binding its promoter. Data were from 3 independent experiments. ∗ P < .05. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: DNA Methylation Assay, Binding Assay, Luciferase, Activity Assay, Comparison, Stable Transfection, Expressing, Western Blot, Knockdown

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: MBD2a induces, whereas MBD2c represses, HCC tumorigenesis and CSC properties. A , Sphere formation and limiting dilution assays when overexpressing MBD2a or with HNRNPM depletion, MBD2c in MHCC97H cells. B , The cell proliferation by CCK-8 assays when overexpressing MBD2a or with HNRNPM depletion, MBD2c in MHCC97H cells. C , Cell migration and migration assay when overexpressing MBD2a or with HNRNPM depletion, MBD2c in MHCC97H cells. Data were from 3 independent experiments. ∗ P < .05. D , Colony formation assay when overexpressing MBD2a or with HNRNPM depletion, MBD2c in MHCC97H cells. Data were from 3 independent experiments. ∗ P < .05. E , The cell apotosis by flow cytometry when overexpressing MBD2a or with HNRNPM depletion, MBD2c in MHCC97H cells. Data were from 3 independent experiments. ∗ P < .05. F , The protein expression of HNRNPM, MBD2a, MBD2c when downregulating SOX2, OCT4, and together with overexpressing HNRNPM by Western blot experiments. G , The in vivo effects in BALB/c nude mice when overexpressing MBD2a (n = 5) or with HNRNPM depletion (n = 5), MBD2c (n = 5). ns, Non-significant. ∗ P < .05, ∗∗ P < .01. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: CCK-8 Assay, Migration, Colony Assay, Flow Cytometry, Expressing, Western Blot, In Vivo, Comparison

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: The coregulated genes by MBD2a and MBD2c in HCC cells. A-B , Venn diagram of the RNA-seq data showing the genes commonly regulated by MBD2a and MBD2c. C-D , Gene Ontology (GO) enrichment analysis. The top 5 GO terms in the indicated categories with the lowest P values are shown. E , The expression of Snail1, OCT4, SOX2 mRNA, and proteins was measured by qPCR and Western blot in MHCC97H cells expressing shRNAs targeting MBD2a, and in MHCC97H cells stably expressing MBD2c. Data were from 3 independent experiments. ∗ P < .05 as compared with controls. F , The expression of β-catenin by nuclear/cytoplasmic protein fractionation and TOP/FOP-flash reporter assays when silencing MBD2a or overexpressing MBD2c. Data were from 3 independent experiments. ∗ P < .05; ∗∗ P < .01 by the Student t test. G , The expression of Snail1, OCT4, SOX2 mRNA, and proteins was measured by qPCR and Western blot in MHCC97H cells expressing HNRNPM and shRNA targeting MBD2a. Data were from 3 independent experiments. ∗∗∗ P < .001 as compared with controls; ns, Not significant; P > .05. H , The expression of β-catenin by nuclear/cytoplasmic protein fractionation and TOP/FOP-flash reporter assays when overexpressing HNRNPM and silencing MBD2a. Data were from 3 independent experiments. ∗∗ P < .01. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: RNA Sequencing, Expressing, Western Blot, Stable Transfection, Fractionation, shRNA, Comparison

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: A , The relative expression of MBD2a, MBD2c in fetal liver, adult liver, HCC, and adjacent noncancerous liver tissues. ∗∗∗ P < .001 by the Student t test. B-C , The Kaplan-Meier analyses of the correlations between MBD2a ( B ), MBD2c ( C ) level and overall survival of n = 100 patients with HCC. The median MBD2a or MBD2c level was used as the cutoff. D , The multivariate analysis for MBD2a and MBD2c in patients with HCC. E , The correlation analysis between HNRNPM and MBD2a in patients with HCC (n = 30) by IHC experiments. F , FZD3 and HNRNPM expression in protein levels in HCC tissues with strong or weak HNRNPM staining intensity. The median HNRNPM staining intensity was used as the cutoff (n = 60 HCC tissues). ∗∗∗ P < .001. G-H , The correlation between the expression of HNRNPM and FZD3 ( G ), β-catenin ( H ) from TCGA datasets.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Expressing, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: The effects of HNRNPM-specific ASO for HCC in vivo and in vitro. A , The expression of HNRNPM was significantly correlated with MBD2a. B , The IC50 of ASO-2 for MHCC97H cells. C , The protein expression of HNRNPM, MBD2a, FZD3, OCT4, SOX2, and β-catenin related assays when treated with ASO-2 in HCC cells. Data were from 3 independent experiments. ∗ P < .05 by the Student t test. D , The CSC markers expression by ASO treatment. E , The CCK-8 experiment when treated with HNRNPM-specific ASO in MHCC97H cells. F , Sphere formation and limiting dilution assays when treated with HNRNPM-specific ASO in MHCC97H cells. Data were from 3 independent experiments. ∗ P < .05 by the Student t test. G , Limiting dilution assays when treated with HNRNPM-specific ASO in MHCC97H cells. H , Colony formation assay when treated with HNRNPM-specific ASO in MHCC97H cells. Data were from 3 independent experiments. ∗ P < .05. I-J , Invasion assay ( I ) and cell migration ( J ) and when treated with HNRNPM-specific ASO in MHCC97H cells. Data were from 3 independent experiments. ∗ P < .05 by the Student t test. K , The HCC cell apoptosis changes by ASO treatment. Data were from 3 experiments. ∗∗ P < .01 by the Student t test. L , The schematic diagram of ASO-2 treating nude mice when inoculating the tumor cells. M , The effects of HNRNPM-specific ASO when treated ASO I.P by 25 mg/kg (n = 5). ∗∗∗ P < .001 by the Student t test. N ,. The HNRNPM expression in tumors when treating HNRNPM-ASO by IHC experiments.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: In Vivo, In Vitro, Expressing, CCK-8 Assay, Colony Assay, Invasion Assay, Migration

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: Expression of HNRNPM correlated with immune checkpoint in human HCC. A-E , Expression correlation between HNRNPM and immune checkpoint gene RNA amounts in the TCGA HCC database, n = 370, HNRNPM (HNRNPM), PD-L1 (CD274), B7-H3 (CD276), B7-H4 (VTCN1), LAG-3 (LAG3), and TIM-3 (HAVCR2). B , Pearson correlation analysis of HNRNPM and CD276 immune checkpoint expressions in human HCC tissue microarray based on the IHC results, n = 240.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Expressing, Microarray

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: HNRNPM inhibition curbs immune escape and enhances PD-1 blockade by promoting CD8+ T cells activation phenotype. A , Schematic diagram of Hep1-6-OVA cells co-cultured with OTI cells. B , The flow cytometry analysis of IFN-γ+ or granzyme B+ CD8+ T cells between control and shHNRNPM groups. Data were from 3 independent experiments. ∗∗∗ P < .001. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test. C , Schematic diagram of ASO and anti-PD-1 therapy in C57/BJ6 mice. D , Tumor inhibition by IgG (n = 6), HNRNPM-ASO (n = 6), anti-PD-1 (n = 6), or combination therapy (n = 6) in C57/BJ6 mice. ∗ P < .05. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test. E , Survival analysis of IgG (n = 6), HNRNPM-ASO (n = 6), anti-PD-1 (n = 6), or combination therapy (n = 6) in C57/BJ6 mice. ∗ P < .05. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test. F , The profiles of immune cells in tumors by HNRNPM-ASO, anti-PD-1 or combination therapy. G , CD8+ T cells infiltration in HNRNPM-ASO, anti-PD-1 or combination therapy groups. ∗∗ P < .01. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test. H , The changes of Treg, IFNG+, GMZB+ CD8+ T cells in control, HNRNPM-ASO, anti-PD-1 or combination therapy groups in tumor-bearing C57/BJ6 mice. ∗∗∗ P < .001. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test. I , The immune cells infiltration landscape of spleen in control, HNRNPM-ASO, anti-PD-1, or combination therapy groups in tumor-bearing C57/BJ6 mice. J , The mice weight between controls and HNRNPM-ASO group. ns , Non-significant. K , The relative expression of β-catenin in HNRNPM-ASO, anti-PD-1, or combination therapy groups. ∗∗∗ P < .001. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test. L-M , The distribution of CTNNB1 mutation in PD-1 responders or non-responders. N , The study model diagram.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Inhibition, Activation Assay, Cell Culture, Flow Cytometry, Control, Comparison, Expressing, Mutagenesis

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: List of Antibodies Used in This Research

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques:

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: List of Primers Sequences and shRNA Sequences Used in this Research

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: shRNA, Sequencing, Control, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: A Monoclonal Anti-HMGB1 Antibody Attenuates Neurodegeneration in an Experimental Animal Model of Glaucoma

doi: 10.3390/ijms23084107

Figure Lengend Snippet: Quantification of the retinal nerve fiber layer thickness (RNFLT). To quantify the damage to the retina caused by the chronically elevated IOP in vivo, the retina was examined by optical coherence tomography (OCT). This allows retinal cross-sections to be imaged. The retinal nerve fiber layer was defined and quantified using a semi-automatic segmentation algorithm. For quantification, a 12° diameter circular B-scan was acquired. The Heidelberg Eye Explorer software divides the retina into the main sectors, superior (S), temporal (T), inferior (I), and nasal (N), as well as into the intermediate sectors, temporal superior (TS), temporal inferior (TI), nasal inferior (NI), and nasal superior (NS) ( A ). The scheme shows the interventions performed during the study on the eye, episcleral vein occlusion (EVO), and intravitreal injection (IVI) (scheme modified from ). The mean RNFLT of the whole B-scan was used for follow-up quantification ( B ). Further quantification of the RNFLT at week 10 was performed for the individual sectors ( C–H ). Legend: black (IgG OS), blue (HMGB1 OS), light grey (IgG OD), dark grey (HMGB1 OD). * p < 0.05, ** p < 0.01, *** p < 0.001, not significant (ns), one-way ANOVA, Tukey’s HSD post hoc test.

Article Snippet: The treatment group (HMGB1 OS, n = 6) received 50 μg of a mouse-derived anti-HMGB1 antibody (1F3, NBP2-25148, Novus Biologicals, Littleton, CO, USA).

Techniques: In Vivo, Tomography, Software, Injection, Modification

Journal: International Journal of Molecular Sciences

Article Title: A Monoclonal Anti-HMGB1 Antibody Attenuates Neurodegeneration in an Experimental Animal Model of Glaucoma

doi: 10.3390/ijms23084107

Figure Lengend Snippet: Summary of OCT analysis including RNFLTs of the IgG OS and HMGB1 groups and their differences.

Article Snippet: The treatment group (HMGB1 OS, n = 6) received 50 μg of a mouse-derived anti-HMGB1 antibody (1F3, NBP2-25148, Novus Biologicals, Littleton, CO, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: A Monoclonal Anti-HMGB1 Antibody Attenuates Neurodegeneration in an Experimental Animal Model of Glaucoma

doi: 10.3390/ijms23084107

Figure Lengend Snippet: Ganzfeld electroretinogram (ERG) pattern and quantification of B-wave and PhNR amplitude at week 0 and week 10. ERG recordings were acquired at different time points. The mean values of the ERG recordings of the individual animals were plotted for the time points week 0 (before EVO) and week 10 after EVO ( A – D ). The flash intensity 1.37 log 10 cd·s·m −2 was used for the ERG recordings shown. The B-wave amplitude (*) and the amplitude of the photopic negative response (PhNR, **) were used for quantification. Quantification was performed separately for the B-wave amplitude of the IgG ( E ) and the HMGB1 group ( F ) as well as for the PhNR amplitude of the IgG ( G ) and the HMGB1 group ( H ). The millivolt decrease was calculated as a percentage difference (Δ = % change).

Article Snippet: The treatment group (HMGB1 OS, n = 6) received 50 μg of a mouse-derived anti-HMGB1 antibody (1F3, NBP2-25148, Novus Biologicals, Littleton, CO, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: A Monoclonal Anti-HMGB1 Antibody Attenuates Neurodegeneration in an Experimental Animal Model of Glaucoma

doi: 10.3390/ijms23084107

Figure Lengend Snippet: Expression pattern of selected retinal proteins analyzed by an antibody microarray. For the analysis, 10 µg of protein lysate was used. This was labeled, and a size exclusion chromatography column removed excess unbound dye and buffers unfavorable for the array incubation. The altered proteins with respective expressions in the elevated-IOP eyes were shown. ** p < 0.01, * p < 0.05, other p values as indicated, Student’s t -test ( A ). The protein expression of the high-mobility group box protein 1 (HMGB1) was examined in the IgG animals (OS, OD) and the antibody-treated HMGB1 animals (OS, OD) ( B – G ).

Article Snippet: The treatment group (HMGB1 OS, n = 6) received 50 μg of a mouse-derived anti-HMGB1 antibody (1F3, NBP2-25148, Novus Biologicals, Littleton, CO, USA).

Techniques: Expressing, Microarray, Labeling, Size-exclusion Chromatography, Incubation

Journal: International Journal of Molecular Sciences

Article Title: A Monoclonal Anti-HMGB1 Antibody Attenuates Neurodegeneration in an Experimental Animal Model of Glaucoma

doi: 10.3390/ijms23084107

Figure Lengend Snippet: Schematic illustration of a proposed mechanism of anti-HMGB1 Ab application. ( A ) Schematic section of the healthy retina. HMGB1 is mainly located in the nucleus. ( B ) Schematic section of glaucomatous retina. HMGB1 translocates from the nucleus to the cytoplasm. Additionally, HMGB1 is secreted by cells and binds its receptors, such as Toll-like receptors (TLRs) or receptor for advanced glycation endproducts (RAGE). As a result, the expression of cytokines such as CXCL8 is induced and leads to an inflammatory response. ( C ) Glaucomatous retina with anti-HMGB1 Abs. Expression and distribution of HMGB1 is comparable to ( B ). By application of anti-HMGB1 Ab, HMGB1 is mainly captured by the Ab, which suppresses binding to TLRs or RAGE. Thereby, signaling through these receptors is reduced and thus the expression of inflammatory cytokines such as CXCL8 is also reduced, consequently reducing the inflammatory response. Abbreviations: RNFL—retinal nerve fiber layer, GCL—ganglion cell layer, IPL—inner plexiform layer, INL—inner nuclear layer, OPL—outer plexiform layer, CXCL8—C-X-C motif chemokine ligand 8/ interleukin 8 (IL-8). ( D ) Legend for subfigures ( A – C ). This figure was created with BioRender.com.

Article Snippet: The treatment group (HMGB1 OS, n = 6) received 50 μg of a mouse-derived anti-HMGB1 antibody (1F3, NBP2-25148, Novus Biologicals, Littleton, CO, USA).

Techniques: Expressing, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: A Monoclonal Anti-HMGB1 Antibody Attenuates Neurodegeneration in an Experimental Animal Model of Glaucoma

doi: 10.3390/ijms23084107

Figure Lengend Snippet: Antibodies used for antibody-based microarray.

Article Snippet: The treatment group (HMGB1 OS, n = 6) received 50 μg of a mouse-derived anti-HMGB1 antibody (1F3, NBP2-25148, Novus Biologicals, Littleton, CO, USA).

Techniques: Microarray

Journal: Cell

Article Title: Multi-level Proteomics Identifies CT45 as a Chemosensitivity Mediator and Immunotherapy Target in Ovarian Cancer

doi: 10.1016/j.cell.2018.08.065

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: A complete list of cell lines can be found in the . table ft1 table-wrap mode="anchored" t5 caption a7 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal Ki-CT45-2 antibody Dr. Hans-Jürgen Heidebrecht (Kiel, Germany) N/A Mouse monoclonal anti-HLA-ABC antibodies W6/32 Thermo Fisher Scientific #MA1-19027; RRID: AB_1076699 Rabbit monoclonal anti-γH2AX Cell Signaling #9718; RRID: AB_2118009 Rabbit polyclonal anti-Cleaved CASP-3 Cell Signaling #9661; RRID: AB_2341188 Rabbit anti-IgG-HRP Cell Signaling #7074; RRID: AB_2099233 Mouse anti-IgG-HRP Cell Signaling #7076; RRID: AB_330924 Rabbit polyclonal anti-PPP4C Bethyl #A300-835A; RRID: AB_597901 Rabbit polyclonal anti-SMEK2 Bethyl #A300-842A; RRID: AB_597905 Rabbit polyclonal anti-KAP1 Bethyl #A300-274A; RRID: AB_185559 Mouse monoclonal anti-Actin Sigma #A5441; RRID: AB_476744 Rabbit polyclonal anti-CT45A Sigma #SAB1301842 Rabbit polyclonal anti-PPP4R2 Sigma #HPA034695; RRID: AB_10671231 Rabbit polyclonal anti-PPP4R3α Sigma #HPA002568; RRID: AB_1079207 Rabbit monoclonal anti-FANCD2 Abcam #ab108928; RRID: AB_10862535 Mouse monoclonal anti-γH2AX Life Technologies #MA1-2022; RRID: AB_559491 Mouse monoclonal anti-V5 Life Technologies #MA5-15253; RRID: AB_10977225 Mouse monoclonal anti-GST Abcam #3G10/1B3; RRID: AB_524046 Biological Samples Ovarian cancer specimens, Tables S1 and S2 This paper N/A Tumor infiltrating T cells This paper N/A Chemicals, Peptides, and Recombinant Proteins Carboplatin Hospira N/A Growth factor reduced Matrigel BD Biosciences (Rockville, MD) 356231 Synthetic HLA-I peptides of CT45 Core facility, Max Planck Institute of Biochemistry, Martinsried, Germany N/A MHC class-I tetramers NIH Tetramer Core Facility N/A Recombinant PP4R3β Dr. Jakob Nilsson (University of Copenhagen, Denmark) N/A Recombinant GST-CDC20 Dr. Jakob Nilsson (University of Copenhagen, Denmark) N/A Critical Commercial Assays Ser/Thr phosphatase assay kit Millipore #17-127 Deposited Data Raw and analyzed data This paper http://proteomecentral.proteomexchange.org/cgi/GetDataset (dataset identifier PXD010372) Patient information Tables S1 and S2 This paper N/A Ovarian cancer proteome data, Table S3 This paper N/A Patient information of tissue microarray, Table S4 This paper N/A HLA-I peptidomes of cell lines, Table S5 This paper N/A RNaseq analysis of 37 tissues Uhlén et al., 2015 https://www.proteinatlas.org/about/download RNAseq expression analysis of HGSOC and across cancers TCGA IlluminaHiseq BC and HiSeqV2_PANCAN Experimental Models: Cell Lines SKOV3ip1, female origin.

Techniques: Recombinant, Phosphatase Assay, Microarray, Expressing, Plasmid Preparation, Sequencing, Software

Journal: Evolution & development

Article Title: Abundant genetic variation in transcript level during early Drosophila development

doi: 10.1111/j.1525-142X.2008.00281.x

Figure Lengend Snippet: Maternal and zygotic gene expression

Article Snippet: Found on Affymetrix Microarray Expressed Varying among lines Varying over time Increasing Declining Changes during development 2089 (AST7 * ) 2065 ** (ST4) 1215 (ST5) 950 (ST6) 698 (ST7) 224 (ST8) 491 (ST9) Strictly maternal 27 (AST12) 27 (ST10) 21 (ST11) 21 (ST12) 19 (ST13) 0 19 (ST14) Maternal 49 (AST13) 50 (ST15) 41 (ST16) 35 (ST17) 22 (ST18) 2 (ST19) 20 (ST20) Strictly Zygotic 53 (AST19) 57 (ST27) 29 (ST28) 25 (ST29) 22 (ST30) 19 (ST31) 3 (ST32) Zygotic 532 (AST18) 514 (ST21) 176 (ST22) 141 (ST23) 127 (ST24) 89 (ST25) 38 (ST26) Open in a separate window * AST stands for Supplementary Table in Arbeitman et al. 2002 ; and ST is for the Supplementary Table in this article.

Techniques: Microarray