Journal: International journal of molecular medicine

Article Title: Nrf2 activation diminishes during adipocyte differentiation of ST2 cells.

doi: 10.3892/ijmm.2011.761

Figure Lengend Snippet: Figure 1. The intracellular glutathione redox potential becomes more oxidized during adipocyte differentiation of ST2 cells. ST2 cells stably expressing Grx1- roGFP2 were induced to differentiate into adipocytes. Every 24 h the cells were excited with 390 and 480 nm lasers and the ratio of emissions in the green channel (535 nm) was calculated. The graph represents the mean ± SD. Three independent experiments were performed, and in each experiment 24 technical replicates were used for each timepoint. *p<0.0001 compared to day 0; †p<0.001; ††p<0.0001.

Article Snippet: ST2 cells are a cloned stromal-cell line from mouse bone marrow; obtained from DSMZ (German Collection of Microorganisms and Cell Cultures).

Techniques: Stable Transfection, Expressing

Journal: International journal of molecular medicine

Article Title: Nrf2 activation diminishes during adipocyte differentiation of ST2 cells.

doi: 10.3892/ijmm.2011.761

Figure Lengend Snippet: Figure 2. Nuclear Nrf2 protein abundance decreases in ST2 cells during adi- pocyte differentiation. ST2 cells were induced to differentiate into adipocytes. Nuclear protein extracts were prepared immediately before the initiation of dif- ferentiation (day 0) and every 24 h thereafter until day 5. Nrf2 expression was assayed by Western immunoblotting. The nuclear abundance of the transcrip- tion factor SP-1 was employed as a loading control. The image is representative of 3 independent experiments. The graph represents the mean ± SD. *p<0.0001 compared to day 0. †p<0.05; ††p<0.0001.

Article Snippet: ST2 cells are a cloned stromal-cell line from mouse bone marrow; obtained from DSMZ (German Collection of Microorganisms and Cell Cultures).

Techniques: Quantitative Proteomics, Expressing, Western Blot, Control

Journal: International journal of molecular medicine

Article Title: Nrf2 activation diminishes during adipocyte differentiation of ST2 cells.

doi: 10.3892/ijmm.2011.761

Figure Lengend Snippet: Figure 3. Nrf2 binding activity decreases during adipocyte differentiation of ST2 cells. ST2 cells were induced to differentiate into adipocytes. Nuclear protein extracts were prepared before the initiation of differentiation (day 0) and every 24 h thereafter until day 5, and were used for EMSAs with an ARE- containing oligonucleotide as a radiolabelled probe. The image is representative of 3 independent experiments. Binding competition experiments performed with ST2 nuclear extracts at day 0 (0d) of differentiation (maximal Nrf2 DNA binding activity) in the presence of a 10-, 50-, or 100-fold molar excess of unlabeled ARE-wt or ARE-mut oligonucleotides. The image is representative of 3 independent experiments.

Article Snippet: ST2 cells are a cloned stromal-cell line from mouse bone marrow; obtained from DSMZ (German Collection of Microorganisms and Cell Cultures).

Techniques: Binding Assay, Activity Assay

Journal: International journal of molecular medicine

Article Title: Nrf2 activation diminishes during adipocyte differentiation of ST2 cells.

doi: 10.3892/ijmm.2011.761

Figure Lengend Snippet: Figure 4. ARE-mediated transcription is reduced during differentiation of ARE-ST2 cells into adipocytes. ARE-ST2 cells were induced to differentiate into adipocytes and luciferase measurements were performed every 24 h until day 5 of differentiation. Luciferase activity was normalized to total protein concentration. The graph represents the means ± SD. Three independent exper- iments were performed, and in each experiment 8 technical replicates were used for each timepoint. *p<0.0001 compared to day 0; †p<0.05; ††p<0.0001.

Article Snippet: ST2 cells are a cloned stromal-cell line from mouse bone marrow; obtained from DSMZ (German Collection of Microorganisms and Cell Cultures).

Techniques: Luciferase, Activity Assay, Protein Concentration

Journal: eLife

Article Title: RANK + TLR2 + myeloid subpopulation converts autoimmune to joint destruction in rheumatoid arthritis

doi: 10.7554/eLife.85553

Figure Lengend Snippet: ( A ) Dimensional reduction projection of monocyte and macrophage cells expressing RANK, TRAP, and/or TLR2 from all sample groups onto two dimensions using principal components, depicting values of Slingshot pseudotime followed by gene expression levels of St3gal4, Tlr2, Tnfrsf11a, and Acp5 . ( B ) A heat map of dynamic expression of St3gal4, Tlr2, Tnfrsf11a, and Acp5 in monocyte and macrophage cells expressing RANK, TRAP, and/or TLR2 from all sample groups. Pseudotime values and annotated cell types are depicted in the color-coded bars atop the heat map. ( C ) Representative images of TRAP staining and immunofluorescence co-staining for RANK (red), TLR2 (green), and DAPI (blue) staining for nuclei for bone marrow macrophages cultured with macrophage colony stimulating factor (M-CSF) ( + ng/ml) and RANKL (200 ng/ml) for 0, 1, 3, and 5 days. Scale bar, 0.1 mm. ( D ) Quantitative analysis of the percentage of TRAP + cells in bone marrow macrophages at different time points (n=4 or 5, one-way ANOVA with Tukey’s multiple comparisons test). ( E–G ) Quantitative analysis of the mean intensity of RANK and TLR2 positive fluorescence, and the percentage of RANK + TLR2 + positive cells in bone marrow macrophages at different time points (n=5 or 6, one-way ANOVA with Tukey’s multiple comparisons test). All data are means ± SD. N.S=No Significant difference, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: The sections were incubated with primary antibodies against RANK (1:100, ab13918, Abcam), TLR2 (1:100, mAb12276, Cell Signaling), Maackia Amurensis Lectin (1:100, B-1265–1, Vector Laboratories), ST3GAL4 (1:100, 13546–1-AP, Proteintech), c-Fos (1:100, mAb2250, Cell Signaling), or ST3GAL1 (1:50, PA5-21721, Thermo Fisher Scientific) overnight at 4 °C.

Techniques: Expressing, Gene Expression, Staining, Immunofluorescence, Cell Culture, Fluorescence

Journal: eLife

Article Title: RANK + TLR2 + myeloid subpopulation converts autoimmune to joint destruction in rheumatoid arthritis

doi: 10.7554/eLife.85553

Figure Lengend Snippet: ( A ) Representative histograms of sialic acid α(2,3)-modified cells in the bone marrow of DBA control mice and CIA mice 2 months after the first immunization. ( B ) Quantitative analysis of the frequency of sialic acid α(2,3)-positive cells in DBA mice and CIA mice (n=4, t -test). ( C ) Representative images of the biotin fluorescence staining for sialic acid α(2,3) (green) and of DAPI staining (blue) of the knee joint sections from DBA mice and CIA mice 2 months after the first immunization. Scale bar, 0.5 mm. ( D ) Quantitative analysis of the mean intensity of sialic acid α(2,3)-positive fluorescence of the images represented in ( C ) (n=6, t -test). ( E ) Heat maps of dynamic expression of marker genes Tlr2, Tnfrsf11a, Acp5, sialyltransferases, and Fos in the RANK + TLR2 − myeloid cells (top) and RANK + TLR2 + myeloid cells (bottom). ( F ) Representative images of the immunofluorescence staining for c-Fos (green) of the knee joint section from DBA mice and CIA mice 2 months after the first immunization. Scale bar, 0.5 mm. ( G ) Quantitative analysis of the mean intensity of c-Fos-positive fluorescence in ( F ) (n=5, t -test). ( H–I ) The mRNA expression changes of c-Fos in the knee joint tissue of CIA and DBA mice ( H ) or in the RANK + TLR2 − and RANK + TLR2 + monocytes ( I ) (n=3, t -test). ( J ) Representative images of the immunofluorescence co-staining for RANK (red) and ST3GAL4 (green) of the knee joint sections from DBA mice and CIA mice 2 months after the first immunization. ( K ) Quantitative analysis of the RANK + ST3GAL4 + cells in ( J ) (n=5, t -test). ( L ) The electrophoresis of CHIP assay for the binding sites of c-Fos in the promoter sequence of the St3gal4 gene. ( M ) The diagram of different locations and sequences of the putative binding sites for c-Fos in the promoter of St3gal4 gene. *p<0.05, ***p<0.001, ****p<0.0001. Figure 6—source data 1. Gel blot of CHIP assay of St3gal4 gene expression.

Article Snippet: The sections were incubated with primary antibodies against RANK (1:100, ab13918, Abcam), TLR2 (1:100, mAb12276, Cell Signaling), Maackia Amurensis Lectin (1:100, B-1265–1, Vector Laboratories), ST3GAL4 (1:100, 13546–1-AP, Proteintech), c-Fos (1:100, mAb2250, Cell Signaling), or ST3GAL1 (1:50, PA5-21721, Thermo Fisher Scientific) overnight at 4 °C.

Techniques: Modification, Control, Fluorescence, Staining, Expressing, Marker, Immunofluorescence, Electrophoresis, Binding Assay, Sequencing, Western Blot, Gene Expression

Journal: Oncotarget

Article Title: MMP3-mediated tumor progression is controlled transcriptionally by a novel IRF8-MMP3 interaction.

doi: 10.18632/oncotarget.3897

Figure Lengend Snippet: Figure 2: IRF8 regulates MMP3 promoter activity in conjunction with PU.1. A. Chromatin immunoprecipitation (ChIP) followed by RT-PCR on the indicated cell lines to determine binding of IRF8 and PU.1 to the putative consensus motif of the mouse MMP3 promoter (-1137 base pairs upstream of the TSS). Data represent one of two independent experiments. B. ChIP followed by quantitative PCR on the indicated cell line to determine enrichment of IRF8 or PU.1 at the putative consensus motif. C. Transfection assays using the CMS4 cell line to measure MMP3 promoter activity in the presence of cDNA encoding full-length IRF8, PU.1 cDNA or both (upper panel) vs. the corresponding empty vector control plasmids (lower panel). D. EMSA using CMS4-SC lysates after incubation with or without the indicated 32P-labeled oligonucleotide probe. Data are representative of two separate experiments.

Article Snippet: In separate experiments, the parental CMS4 cell line was transfected with an empty vector control (VC; pcDNA3.1+ ) or cDNA encoding the fulllength mouse MMP3 gene (Origene, Rockville, MD) to generate MMP3-overexpressing CMS4 cells (termed, CMS4-MMP3hi), which were maintained and propagated under G418 (Geneticin) selection.

Techniques: Activity Assay, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Control, Incubation, Labeling

Journal: Oncotarget

Article Title: MMP3-mediated tumor progression is controlled transcriptionally by a novel IRF8-MMP3 interaction.

doi: 10.18632/oncotarget.3897

Figure Lengend Snippet: Figure 4: Modulating tumor-derived MMP3 levels alters CMS4 tumor growth. A. MMP3 expression in parental CMS4 cells after stable MMP3 knockdown vs. SC, as shown by qPCR. B. Tumor growth illustrated for the indicated cell line (n = 16 mice per tumor cell line). C. Ectopic overexpression of MMP3 in CMS4 cells as shown at RNA (qPCR, upper panel) or protein (ELISA, lower panel) levels. D. Tumor growth in wild-type mice (n = 15 per tumor cell line) or SCID mice (n = 5 per tumor cell line). *P < 0.05.

Article Snippet: In separate experiments, the parental CMS4 cell line was transfected with an empty vector control (VC; pcDNA3.1+ ) or cDNA encoding the fulllength mouse MMP3 gene (Origene, Rockville, MD) to generate MMP3-overexpressing CMS4 cells (termed, CMS4-MMP3hi), which were maintained and propagated under G418 (Geneticin) selection.

Techniques: Derivative Assay, Expressing, Knockdown, Over Expression, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: MMP3-mediated tumor progression is controlled transcriptionally by a novel IRF8-MMP3 interaction.

doi: 10.18632/oncotarget.3897

Figure Lengend Snippet: Figure 5: Modulating tumor-derived MMP3 levels alters CMS4-met.sel tumor growth. A. Comparison of basal MMP3 expression between CMS4 and CMS4.met.sel at RNA (qPCR, left panel) or protein (ELISA, right panel) levels. B. Knockdown of MMP3 in CMS4.met.sel cells compared to the SC population as shown at RNA (qPCR, left panel) or protein (ELISA, right panel) levels. C. Tumor growth in wild-type mice (n = 18 per tumor cell line; left) or SCID mice (n = 5 per tumor cell line; right). D. Experimental lung metastases as shown by H&E-stained lung tissue sections (left), followed by quantification of approximate volume of lung occupied by tumor (right). Arrows indicate examples of lung tumor nodules. E. MMP3 levels in tumor explants at different time points post-implantation, as measured by qPCR. *P < 0.05 (experimental replicates).

Article Snippet: In separate experiments, the parental CMS4 cell line was transfected with an empty vector control (VC; pcDNA3.1+ ) or cDNA encoding the fulllength mouse MMP3 gene (Origene, Rockville, MD) to generate MMP3-overexpressing CMS4 cells (termed, CMS4-MMP3hi), which were maintained and propagated under G418 (Geneticin) selection.

Techniques: Derivative Assay, Comparison, Expressing, Enzyme-linked Immunosorbent Assay, Knockdown, Staining

Journal: Oncotarget

Article Title: MMP3-mediated tumor progression is controlled transcriptionally by a novel IRF8-MMP3 interaction.

doi: 10.18632/oncotarget.3897

Figure Lengend Snippet: Figure 6: Modulating tumor-derived MMP3 levels alters 4T1 tumor growth. A. Endogenous MMP3 production levels in 4T1 cells compared to CMS4.met.sel cells, as shown by ELISA. B. Knockdown of MMP3 in 4T1 cells compared to SC population as shown at RNA (qPCR, left panel) or protein (ELISA, right panel) levels. C. Tumor growth in wild-type mice (n = 18 for each tumor cell line). D. Experimental lung metastases as shown by H&E-stained lung tissue sections (upper) followed by histologic quantification of tumor nodules (lower). Arrows indicate examples of lung tumor nodules (n = 4 per group). *P < 0.05.

Article Snippet: In separate experiments, the parental CMS4 cell line was transfected with an empty vector control (VC; pcDNA3.1+ ) or cDNA encoding the fulllength mouse MMP3 gene (Origene, Rockville, MD) to generate MMP3-overexpressing CMS4 cells (termed, CMS4-MMP3hi), which were maintained and propagated under G418 (Geneticin) selection.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Knockdown, Staining

Journal: Oncotarget

Article Title: MMP3-mediated tumor progression is controlled transcriptionally by a novel IRF8-MMP3 interaction.

doi: 10.18632/oncotarget.3897

Figure Lengend Snippet: Figure 7: Tumor-derived MMP3 impacts spontaneous metastasis. A. Quantification of spontaneous lung metastasis of the indicated 4T1 cell population with primary tumors left intact. Data collected at endpoint when mice harbored similar primary tumor volumes (4T1-SC: 1794 ± 147 mm3; 4T1-MMP3lo: 1798 ± 143 mm3) and quantified by lung weight or lesion count of H&E-stained lung tissue sections. Each data point represents a single mouse. B. After surgical removal of the primary tumor (i.e., 4T1-SC: 113 ± 9.2 mm3, 4T1-MMP3lo: 126 ± 5.5 mm3), mice were followed for survival based on the appearance of signs/symptoms of morbidity (n = 21 mice per tumor cell line). C. Gene expression profiling of the indicated 4T1 tumor cell population in biologic triplicates, as shown by heat-map of differentially expressed genes (n = 58; > 2-fold up or down; P < 0.01). D. Data in C regrouped based on gene ontogeny analysis. Microarray studies were performed using the MouseWG-6 whole-genome gene expression array.

Article Snippet: In separate experiments, the parental CMS4 cell line was transfected with an empty vector control (VC; pcDNA3.1+ ) or cDNA encoding the fulllength mouse MMP3 gene (Origene, Rockville, MD) to generate MMP3-overexpressing CMS4 cells (termed, CMS4-MMP3hi), which were maintained and propagated under G418 (Geneticin) selection.

Techniques: Derivative Assay, Staining, Gene Expression, Microarray

Journal: Oncotarget

Article Title: MMP3-mediated tumor progression is controlled transcriptionally by a novel IRF8-MMP3 interaction.

doi: 10.18632/oncotarget.3897

Figure Lengend Snippet: Figure 8: Tumor-derived MMP3 modulates invasive capacity and MMP9 activity in vitro. A. Invasion capacity of the indicated tumor cell line (SC vs. MMP3lo), as determined by basement membrane penetration in two-dimensional invasion chamber assays (first bar of each pair set at 1). B. Assays in A performed in the absence or presence of the pan MMP inhibitor GM- 6001 for the CMS4.met.sel model. C&D. MMP9 activity, as assessed by zymography. Upper, representative gels for the indicated tumor cell line pairs (SC vs. knockdown), which shows in situ digestion of gelatin within the gel matrix. Lower, quantification for each cell line; data obtained by densitometry of each band. Each value was then divided by the respective sample-volume to yield a measurement of activity per μl of cell-free supernatant. The values for each cell line were then pooled across three separate experiments and reported as mean ± SEM.

Article Snippet: In separate experiments, the parental CMS4 cell line was transfected with an empty vector control (VC; pcDNA3.1+ ) or cDNA encoding the fulllength mouse MMP3 gene (Origene, Rockville, MD) to generate MMP3-overexpressing CMS4 cells (termed, CMS4-MMP3hi), which were maintained and propagated under G418 (Geneticin) selection.

Techniques: Derivative Assay, Activity Assay, In Vitro, Membrane, Zymography, Knockdown, In Situ

Journal: BMC Cancer

Article Title: The DNA methylation profile of liver tumors in C3H mice and identification of differentially methylated regions involved in the regulation of tumorigenic genes

doi: 10.1186/s12885-018-4221-0

Figure Lengend Snippet: Shortlist of the selected genes a

Article Snippet: After attaching to the dish, cells were incubated in a culture medium containing 5-aza-dC for 72 h. For overexpression study, mouse ORF clones Mst1r (NM_001287261) and Slpi (NM_011414), and pCMV6-Kan/Neo (pCMV6KN) as an empty vector were purchased from ORIGENE Inc. (MD, USA).

Techniques: Methylation, Gene Expression, Protease Inhibitor, Translocation Assay, Membrane, Binding Assay, Derivative Assay

Journal: BMC Cancer

Article Title: The DNA methylation profile of liver tumors in C3H mice and identification of differentially methylated regions involved in the regulation of tumorigenic genes

doi: 10.1186/s12885-018-4221-0

Figure Lengend Snippet: Mst1r associated with hypo-DMR is up-regulated in the liver tumor tissues of C3H mice. a Schematic view of the Mst1r locus. Methylation levels of CpG sites were visualized using Integrative Genomics Viewer (IGV) software. The position of TSS, DMR and the positions assessed by bisulfite sequencing (BS-1 and BS-2) were indicated. The magnified view indicates 8 CpG positions in the DMR and predicted transcription factor binding sites. b The average methylation values of the CpG sites in normal and tumor tissues. The CpG sites with ≥10 reads on both strands (≥20 reads in total) are indicated. Statistical significance between normal and tumor tissue was analyzed by Student’s t-test. **, *** Significantly different at p < 0.01 and 0.001, respectively. c Validation of RRBS data for normal and tumor liver tissues of C3H mice by bisulfite sequencing. Methylated and unmethylated cytosine are shown as ● and ○ respectively. The numbers above the circles indicate the position of CpG shown in Fig. . d Validation of gene expression levels of Mst1r by real-time PCR in the normal ( n = 6) and tumor tissues ( n = 11) of C3H mice. The expression of Mst1r is normalized to the expression of rRNA . Statistical significance between the two groups was analyzed by Student’s t-test. *** Significantly different at p < 0.001

Article Snippet: After attaching to the dish, cells were incubated in a culture medium containing 5-aza-dC for 72 h. For overexpression study, mouse ORF clones Mst1r (NM_001287261) and Slpi (NM_011414), and pCMV6-Kan/Neo (pCMV6KN) as an empty vector were purchased from ORIGENE Inc. (MD, USA).

Techniques: Methylation, Software, Methylation Sequencing, Binding Assay, Biomarker Discovery, Gene Expression, Real-time Polymerase Chain Reaction, Expressing

Journal: BMC Cancer

Article Title: The DNA methylation profile of liver tumors in C3H mice and identification of differentially methylated regions involved in the regulation of tumorigenic genes

doi: 10.1186/s12885-018-4221-0

Figure Lengend Snippet: Reduced DNA methylation of DMRs of Mst1r, Slpi, and Extl1 after 5-aza-dC treatment are associated with up-regulation of these genes in hepa1c1c7 cells and Hepa1-6 cells. Hepa1-6 cells ( b , d , e ) and Hepa1c1c7 cells ( a , c ) were cultured with 0, 0.1 or 1 μM of 5-aza-dC, and 0, 50 or 100 μM of 5-aza-dC for 72 h, respectively. Left figures: the results of bisulfite sequencing of CpGs detected in the DMRs of liver tumors in C3H mice. ●: methylated cytosine, ○: unmethylated cytosine. Right figures: the expressions of Mst1r, Slpi, and Extl were measured by real-time PCR and normalized to the expression of β-actin or rRNA ( n = 3). Statistical significance was analyzed by one-way ANOVA followed by Turkey-Kramer test as a post hoc comparison. * , ** , *** Significantly different at p < 0.05, 0.01, and 0.001, respectively

Article Snippet: After attaching to the dish, cells were incubated in a culture medium containing 5-aza-dC for 72 h. For overexpression study, mouse ORF clones Mst1r (NM_001287261) and Slpi (NM_011414), and pCMV6-Kan/Neo (pCMV6KN) as an empty vector were purchased from ORIGENE Inc. (MD, USA).

Techniques: DNA Methylation Assay, Cell Culture, Methylation Sequencing, Methylation, Real-time Polymerase Chain Reaction, Expressing, Comparison

Journal: BMC Cancer

Article Title: The DNA methylation profile of liver tumors in C3H mice and identification of differentially methylated regions involved in the regulation of tumorigenic genes

doi: 10.1186/s12885-018-4221-0

Figure Lengend Snippet: Overexpression of Mst1r induces IL33 in Hepa1c1c7 cells. Overexpression of Mst1r ( a ) and upregulation of IL33 by Mst1r overexpression ( b ) in Hepa1c1c7 cells was confirmed by real-time PCR. *** Significantly different at p < 0.001 ( n = 3). ( c ) Confirmation by real-time PCR of upregulation of IL33 in the tumor tissues ( n = 11) compared to the normal tissues ( n = 6) of C3H mice. Statistical significance between the two groups was analyzed by the Student’s t-test. *** Significantly different at p < 0.001

Article Snippet: After attaching to the dish, cells were incubated in a culture medium containing 5-aza-dC for 72 h. For overexpression study, mouse ORF clones Mst1r (NM_001287261) and Slpi (NM_011414), and pCMV6-Kan/Neo (pCMV6KN) as an empty vector were purchased from ORIGENE Inc. (MD, USA).

Techniques: Over Expression, Real-time Polymerase Chain Reaction

Journal: BMC Cancer

Article Title: The DNA methylation profile of liver tumors in C3H mice and identification of differentially methylated regions involved in the regulation of tumorigenic genes

doi: 10.1186/s12885-018-4221-0

Figure Lengend Snippet: Human database searches showed hypomethylation of downstream regions of MST1R and SLPI with upregulation of their expressions. RNA-seq dataset and DNA methylation dataset of 41 paired normal and tumor tissues of human livers were downloaded from TCGA. a , b Average gene expression ratio (tumor tissues/normal tissues) was calculated using 41 paired data from TCGA. * , ** Significantly different at P < 0.05, and 0.01, respectively. c The difference in DNA methylation β-value (tumor – normal) for each CpG around TSS was calculated using 41 paired data from TCGA. d The correlation between expressions of MST1R and IL33 in the HCC tissues highly expressing MST1R

Article Snippet: After attaching to the dish, cells were incubated in a culture medium containing 5-aza-dC for 72 h. For overexpression study, mouse ORF clones Mst1r (NM_001287261) and Slpi (NM_011414), and pCMV6-Kan/Neo (pCMV6KN) as an empty vector were purchased from ORIGENE Inc. (MD, USA).

Techniques: RNA Sequencing, DNA Methylation Assay, Gene Expression, Expressing