Journal: JCI Insight

Article Title: Virus-induced RGMa expression drives neurodegeneration in HTLV-1–associated myelopathy

doi: 10.1172/jci.insight.184530

Figure Lengend Snippet: ( A ) The concentration of NF-L (pg/mL) in the supernatant when NB-1 cells were cocultured with HAM-PBMCs ( n = 7) or HD-PBMCs ( n = 7). ( B ) The concentration of NF-L (pg/mL) in the supernatant when NB-1 cells were cocultured with HAM-PBMCs ( n = 9) and with mogamulizumab (antiCCR4) in a dose-dependent manner for 72 hours. ( C ) The comparison of RGMA mRNA gene expression levels using DNA microarray among normal CD4 + T cells (HD CD4 + : n = 4), HAM patient–derived CD4 + T cells (HAM CD4 + : n = 4), ACs ( n = 2), and smoldering/chronic-type-ATL patient–derived ( n = 3) HTLV-1–infected CD4 + T cells (Non-HAM infected CD4 + T cells: n = 5), and acute-type-ATL patient–derived HTLV-1–infected CD4 + T cells (Acute ATL infected cells: n = 3). ( D ) The comparison of the expression levels of the genes associated with the inhibition of neuroregeneration ( OMG , MAG , RTN4 , and WNT5A ) between HD CD4 + ( n = 4) and HAM CD4 + T cells ( n = 4). ( E ) The enrichment levels of H3K27me3 –2916 bp upstream from the TSS of the RGMA gene locus in HD CD4 + ( n = 3), HAM CD4 + ( n = 4), and acute-ATL infected cells ( n = 4). Data are shown as mean ± SD. ** P < 0.01; *** P < 0.001 by unpaired t test ( A and D ) or 1-way ANOVA with Dunnett’s multiple-comparison test ( B , C , and E ). NF-L, neurofilament light chain.

Article Snippet: In the experiment analyzing Tax , HBZ , and RGMA gene expression levels in cultured HAM-PBMCs, mRNA was purified using the Magnetic mRNA isolation kit (New England BioLabs).

Techniques: Concentration Assay, Comparison, Gene Expression, Microarray, Derivative Assay, Infection, Expressing, Inhibition

Journal: JCI Insight

Article Title: Virus-induced RGMa expression drives neurodegeneration in HTLV-1–associated myelopathy

doi: 10.1172/jci.insight.184530

Figure Lengend Snippet: ( A ) The validation of RGMA mRNA gene expression levels using qRT-PCR in HD CD4 + ( n = 6) and HAM CD4 + T cells ( n = 6). ( B ) Expression of RGMa protein in CD3 + CD4 + CCR4 + T cells from HAM-PBMCs. Representative dot plots of CCR4 and normal goat IgG (upper) or RGMa expression (bottom) in CD3 + CD4 + gated cells from HD-PBMCs (left) or HAM-PBMCs (right) cultured for 2 days. ( C ) Graph shows the percentage of RGMa protein–expressing cells in CCR4 – cells or CCR4 + cells in CD3 + CD4 + gated cells from HAM-PBMCs ( n = 8) cultured for 2 days, compared with the isotype control, normal goat IgG. ( D ) Graph shows the percentage of RGMa protein–expressing cells among CD3 + CD4 + CCR4 + gated cells from HD-PBMCs ( n = 5) or HAM-PBMCs ( n = 8) cultured for 2 days. Data are shown as mean ± SD. * P < 0.05; ** P < 0.01 by unpaired t test ( A and D ) or 1-way ANOVA with Dunnett’s multiple-comparison test.

Article Snippet: In the experiment analyzing Tax , HBZ , and RGMA gene expression levels in cultured HAM-PBMCs, mRNA was purified using the Magnetic mRNA isolation kit (New England BioLabs).

Techniques: Biomarker Discovery, Gene Expression, Quantitative RT-PCR, Expressing, Cell Culture, Control, Comparison

Journal: JCI Insight

Article Title: Virus-induced RGMa expression drives neurodegeneration in HTLV-1–associated myelopathy

doi: 10.1172/jci.insight.184530

Figure Lengend Snippet: ( A ) Tax (left), HBZ (middle), and RGMA (right) gene expression levels in cultured HAM-PBMCs ( n = 7) in a time-dependent manner. RPL19 was used as an internal control. ( B ) Tax-dependent RGMA mRNA gene induction in Jurkat cells, which were infected with lentivirus carrying the Tax gene. Top: Tax expression in the Jurkat cells was confirmed by Western blotting. β-Actin was measured as an internal control. Bottom: The induction levels of the RGMA gene were evaluated by qRT-PCR in a time-dependent manner ( n = 3). ( C ) Tax -dependent RGMA mRNA gene induction in JPX9 cells treated with 20 μM CdCl 2 in a time-dependent manner. Tax mRNA (upper) and RGMA mRNA (bottom) were measured by qRT-PCR ( n = 3). GAPDH was measured as an internal control. ( D ) Tax-dependent RGMa protein induction in JPX9 cells treated with 20 μM CdCl 2 for 3 days. Dot plots of Tax and normal goat IgG (upper) or RGMa expression (bottom) in JPX9 cells. JPX9(-), untreated JPX9 cells; 20 μM CdCl 2 JPX9, CdCl 2 -supplemented JPX9 cells. Data are shown as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001 by 1-way ANOVA with Dunnett’s multiple-comparison test ( A ), 2-sided Student’s t test ( B ), or an unpaired t test ( C ). Experiments were performed in triplicate ( B and C ).

Article Snippet: In the experiment analyzing Tax , HBZ , and RGMA gene expression levels in cultured HAM-PBMCs, mRNA was purified using the Magnetic mRNA isolation kit (New England BioLabs).

Techniques: Gene Expression, Cell Culture, Control, Infection, Expressing, Western Blot, Quantitative RT-PCR, Comparison

Journal: Scientific reports

Article Title: ROR2 expression predicts human induced pluripotent stem cell differentiation into neural stem/progenitor cells and GABAergic neurons.

doi: 10.1038/s41598-023-51082-4

Figure Lengend Snippet: Figure 3. ROR2 negatively regulates the differentiation of hiPSCs into NS/PCs. (a) Microarray data of ROR2 expression in 10 hiPSC lines (n = 6, biological replicates). Using one-way ANOVA, a significant difference in ROR2 expression was observed between cell lines (P < 0.0001). (b) ROR2 KD was confirmed using qRT-PCR analysis (n = 3, biological replicates). (c) Western blotting analysis of the total extracts obtained from control and ROR2 KD cells. β-actin was used as a loading control. Molecular weight is indicated as Mr (k). (d) qRT-PCR analysis of undifferentiated hPSC markers, OCT3/4 and LIN28A. Total RNA was isolated from R-2A ROR2 KD cells and R-2A control shRNA cells in the undifferentiated state (n = 3, biological replicates). (e, f) qRT-PCR analysis of NS/PC and astrocyte marker genes in NS/PCs derived from ROR2 KD and control shRNA cells (n = 3, biological replicates). Suspension method (e) and adhesion method (f) are shown. (g) Immunofluorescence staining of PAX6 (red) and DAPI (blue) in control (upper) and ROR2 KD (lower) cells. Scale bars, 100 µm. *P < 0.05, **P < 0.01, ****P < 0.0001 (two-tailed unpaired t-test). Error bars represent mean ± SD.

Article Snippet: Primary antibody dilutions were prepared in Can Get Signal immunoreaction enhancer solution (TOYOBO) as follows: anti-ROR2 antibody (AF2064; R&D Systems) 1:1000, anti-β-actin antibody (A5441; Sigma-Aldrich) 1:2000.

Techniques: Microarray, Expressing, Quantitative RT-PCR, Western Blot, Control, Molecular Weight, Isolation, shRNA, Marker, Derivative Assay, Suspension, Immunofluorescence, Staining, Two Tailed Test

Journal: Scientific reports

Article Title: ROR2 expression predicts human induced pluripotent stem cell differentiation into neural stem/progenitor cells and GABAergic neurons.

doi: 10.1038/s41598-023-51082-4

Figure Lengend Snippet: Figure 5. ROR2 knockdown promotes differentiation into forebrain neurons. (a) Schematic of culture procedures for midbrain neuron differentiation. (b) qRT-PCR analysis of the mRNA levels of the midbrain neuron markers TH, FOXA2, BN1, and mature neuron marker TUBB3. Total RNA was isolated from R-2A ROR2 KD cells and R-2A control shRNA cells that were differentiated into midbrain neurons (day 42, n = 3, biological replicates). (c) Schematic of culture procedures for forebrain neuron differentiation. (d) qRT-PCR analysis of the mRNA levels of the forebrain neuron markers MAP2, GAD1, SLC6A1, and VGLUT1 and the mature neuron marker TUBB3. Total RNA was isolated from R-2A ROR2 KD cells and R-2A control shRNA cells that were differentiated into forebrain neurons (day 42, n = 3, biological replicates). (e) Immunofluorescence staining of MAP2 (left) and GAD1 (right) in control (upper) and ROR2 KD (lower) cell-derived forebrain neuron. Scale bars, 100 µm. *P < 0.05, **P < 0.01, ****P < 0.0001 (two-tailed unpaired t-test). Error bars represent mean ± SD.

Article Snippet: Primary antibody dilutions were prepared in Can Get Signal immunoreaction enhancer solution (TOYOBO) as follows: anti-ROR2 antibody (AF2064; R&D Systems) 1:1000, anti-β-actin antibody (A5441; Sigma-Aldrich) 1:2000.

Techniques: Knockdown, Quantitative RT-PCR, Marker, Isolation, Control, shRNA, Immunofluorescence, Staining, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: ( a ) Immunohistochemistry showing reduced methylated H3K79 levels (H3K79me2) that reflect loss of DOT1L activity in damaged areas from osteoarthritic patients (OA) as compared to their corresponding preserved areas and to cartilage from non-OA patients. Images are representative of images from four different patients. Scale bar, 400 μm. ( b ) Heat maps of differential mRNA expression determined by quantitative PCR in chondrocytes treated with DOT1L inhibitor EPZ-5676 (EPZ) or vehicle (V) from passage 0 (P0) until P2, and from preserved versus damaged areas in OA cartilage. The colour code represents the mean expression level of six and four independent patient samples respectively. ( c ) Immunoblot analysis showing decreased methylated H3K79 levels in mouse articular chondrocytes after intra-articular injection of EPZ into C57Bl/6 wild-type mouse knees. The image is representative of one experiment with protein extracts pooled from two or three mice per condition. Unprocessed original scans of blots are shown in . ( d , e ) C57/Bl6 wild-type mouse knees were injected with EPZ (5 mg kg –1 ) or vehicle and killed after 2 or 4 weeks. Knees were sectioned and stained with Hematoxylin-Safranin O ( d ). Scale bar, 200 μm. Cartilage damage was scored (see Methods section) and is shown in ( e ). One experiment was performed with n =10 and 5. Representative images from the 4 week evaluation are shown. * P <0.05 (two-tailed t -test). Error bars indicate mean±s.e.m.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Immunohistochemistry, Methylation, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Injection, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: ( a ) KEGG pathway enrichment analysis of microarray data obtained from human articular chondrocytes treated with EPZ-5676 or vehicle. Nominal P values by EASE modified Fisher Exact test using the DAVID analysis tool (see Methods section) are shown. n =5 independent patient-derived cell cultures. ( b ) Co-immunoprecipitation (Co-IP) using an anti-DOT1L antibody showing interaction between DOT1L and β -catenin in human articular chondrocytes, that is increased upon Wnt activation by LiCl and disrupted upon DOT1L inhibition. The image is representative of three experiments. ( c ) TOP/FOP reporter assay in human articular chondrocytes after Wnt stimulation by LiCl and DOT1L inhibition by EPZ. Activity is compared to untreated cells (dotted line). n =3 biologically independent experiments. *** P <0.001 by one-way ANOVA. ( d , e ) LEF1 , TCF1 and c-MYC expression measured by quantitative PCR in chondrocytes treated with EPZ-5676 and LiCl ( d ) or in LiCl-treated chondrocytes transfected with siRNA directed against DOT1L or scrambled siRNA (siDOT1L or siSCR, respectively) ( e ). Data are from one experiment with three technical replicates. ( f ) Immunohistochemistry demonstrating increased TCF1 levels in the articular cartilage of C57/Bl6 wild-type mice after injection of EPZ-5676. The images are representative of three different animals. Scale bar, 200 μm.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Microarray, Modification, Derivative Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Activation Assay, Inhibition, Reporter Assay, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Immunohistochemistry, Injection

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: All experiments were performed in healthy human articular chondrocytes: treated as indicated with DOT1L inhibitor EPZ-5676, Wnt activator LiCl, SIRT1 antagonist EX527 or SIRT1 agonist SRT1720; or transfected with DOT1L or scrambled siRNA. All data are presented as mean±s.e.m. ( a ) Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) analysis of DOT1L and methylated H3K79 and ( b ) acetylated H3K9 (H3K9Ac) and methylated H3K4 (H3K4me3) as markers of active transcription on the transcriptional start site (TSS) of Wnt target genes. Data are from two to five experiments. ( c ) Expression levels of TCF1 Wnt target gene measured by quantitative PCR in chondrocytes transfected with indicated specific or scrambled siRNA (siSCR). Data are from one experiment with technical triplicates. ( d ) TCF1 expression measured by quantitative PCR in the presence of SIRT1 agonist and antagonist. Data from two experiments each with technical triplicates. ( e ) Co-IP analysis using the indicated antibodies demonstrating the interaction of DOT1L and SIRT1. The image is a representative image of three biologically independent experiments. ( f ) SIRT1 activity relative to vehicle-treated cells (dotted line). Data are from three biologically independent experiments. * P <0.05, *** P <0.001 by one-way ANOVA. ( g ) ChIP-qPCR analysis of SIRT1, PPARGC1A, GCN5 and EP300 binding on the TCF1 promoter and ( h ) TCF1 expression after siRNA transfection with indicated specific or scrambled siRNA. Data from two biologically independent experiments.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR, Methylation, Expressing, Co-Immunoprecipitation Assay, Activity Assay, Binding Assay

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: ( a – c ) Inactivation of SIRT1 protects against DOT1L inhibitor-induced osteoarthritis: ( a ) C57/Bl6 wild-type mouse knees were injected with DOT1L inhibitor EPZ-5676 (5 mg kg −1 ) and SIRT1 inhibitor EX527 (1.25 mg kg –1 ), or vehicle (V) and killed after 4 weeks. Knees were sectioned and stained with Hematoxylin-Safranin O ( a ). Scale bar, 200 μm. Cartilage damage was scored (see Methods section) and is shown in ( b ). One experiment was performed with n =3 (vehicle), 8 (EPZ) and 10 (EPZ+EX527). * P <0.05 by one-way ANOVA. Error bars indicate mean±s.e.m. ( c ) Immunohistochemistry of TCF1 in the indicated groups. TCF1 levels are increased after EPZ treatment and normalized by additional EX527 treatment. The images are representative of three different animals. Scale bar, 200 μm. ( d , e ) Loss of DOT1L function causes severe growth retardation as demonstrated by skeletal staining ( d ) and histology of the growth plate ( e ) of 4-week-old Dot1l fl/fl ;Col2-Cre −/− (Cre-neg) and Dot1l fl/fl ;Col2-Cre +/− (Dot1l Cart-KO ) mice. ( f , g ) Increased TCF1 levels in Dot1l Cart-KO mice as shown by immunohistochemistry in the indicated mice strains in the articular cartilage ( f ) and growth plate ( g ). The images are representative of three different animals. Scale bar, 200 and 100 μm.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Injection, Staining, Immunohistochemistry

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: Upon Wnt signalling activation, DOT1L-containing complexes bind Wnt target gene chromatin. DOT1L interacts with SIRT1 and inhibits its function, preventing Wnt pathway hyper-activation. When Wnt signalling is activated in the absence of DOT1L function, high SIRT1 activity mediates the recruitment of transcriptional activators to LEF1 and TCF1 genes. High Wnt signalling leads to deleterious downstream effects and loss of cartilage homeostasis.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Activation Assay, Activity Assay

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

doi: 10.3390/cancers14133141

Figure Lengend Snippet: Characterization of CAFs and NFs obtained from clinical surgical tissues from patients with head and neck cancer (HNC) ( a ) Morphological comparisons between CAFs and NFs from a representative HNC case showed that CAFs (right panel) consisted of more cytoplasmic protrusions than NFs (left panel). Photographs were captured at 40× magnification. ( b ) Quantitative PCR (left panel) of the culture medium showed a significantly higher expression of vimentin and α-SMA in CAFs than in NFs. Western blot analysis (right panel) also demonstrated that levels of vimentin and α-SMA were significantly higher in CAFs than in NFs. ( c ) Flow cytometric analysis of the cell surface markers, CD10 and GPR77, showed a marked increase in their expression in CAFs compared to NFs. ( d ) A heat map of the gene microarray of NFs and CAFs showed that there were several differences in the expression profile of the secreted genes. The arrow indicates a marked discrepancy of the relative mRNA levels of CCL11 in CAF compared with NF. ( e ) The RT-PCR (left panel) and ELISA (right panel) analysis showed an increased expression of CCL11 in CAFs compared with that in NFs. ( f ) Western blot analysis showed that the protein level of CCL11 was higher in CAFs than in NFs in cell lysates. ( g ) Western blot analysis showed a higher CCL11 expression in CAF-CM compared to NF-CM. The asterisk indicated a significant difference (*: p < 0.05; **: p < 0.01) between experimental and control groups.

Article Snippet: CAFs and NFs supernatant CCL11concentrations were measured using the CCL11 (human) ELISA kit (R&D system) following the manufacturer’s protocol.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Microarray, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Control

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

doi: 10.3390/cancers14133141

Figure Lengend Snippet: CCL11 produced by CAFs causes increased migration and invasion, and the EMT of HNC cells. ( a ) Comparative analysis of the migration and invasion of HNC cells associated with CCL11. Four test groups were classified for comparative analysis of migration and invasive abilities. FaDu and NPC204 cells cultured with medium containing CAF-induced CCL11 presented greater abilities of migration and invasion, with a statistically significant difference, than three other groups: NF, NF with CCL11, and CAFs treated with CCL11 antibody. The asterisk indicates a significant difference (*: p < 0.05; **: p < 0.01) ( b ) Comparative photographs of the infiltrating behavior of FaDu and NPC204 cells in an organotypic culture in four groups seeded onto a mixture layer containing NFs or CAFs with CCL11 or CCL11 antibody. The arrow(s) indicate infiltration buds from the HNC cells seeded above. ( c ) Representative blots of the EMT-associated markers in FaDu and NPC204 cells, as observed upon Western blotting analysis in five groups, showed that treatment with CAF-conditioned medium or the application of rCCL11 decreased the expression of epithelial-type markers (E-cadherin), and increased the expression of mesenchymal-type markers (fibronectin) and EMT regulators (Snail and Twist). In addition, increased expression of invasion-related MMP2 and MMP9 was also seen in those two groups, compared with other groups. The asterisk indicates a significant difference ( p < 0.05) between experimental and control groups. Results are expressed as mean ± SD.

Article Snippet: CAFs and NFs supernatant CCL11concentrations were measured using the CCL11 (human) ELISA kit (R&D system) following the manufacturer’s protocol.

Techniques: Produced, Migration, Cell Culture, Western Blot, Expressing, Control

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

doi: 10.3390/cancers14133141

Figure Lengend Snippet: Comparative analysis of induction of CSC properties and drug resistance in HNC cells associated with CCL11. ( a ) Four groups were classified for comparative analysis of the ability of sphere formation. Increased ability of sphere formation in two test groups of HNC cells exposed to a CAF medium and the group with treatment of rCCL11 was noted. ( b ) Flow cytometric analysis showed a significant increase in CD44 and CD44/CD24, as well as in CD133 in HNC cells exposed to rCCL11, compared to control HNC cells ( p < 0.05). ( c ) Flow cytometric analysis showed a marked increase in ALDH-1 activity in HNC cells exposed to rCCL11 compared to control HNC cells. ( d ) Western blot analysis showed that CSC-representative markers, Oct-4, Nanog, and Sox-2, were also overexpressed in addition to the increased expression of two important drug resistance genes, ABCG-2 and MDR-1 , in HNC cells exposed to rCCL11. ( e ) Treatment with Cisplatin at 24 h showed a significant increase in chemoresistance in both FaDu and NPC204 cells exposed to rCCL11 compared with control HNC cells. The asterisk indicates a significant difference (*: p < 0.05; **: p < 0.01) between experimental and control groups.

Article Snippet: CAFs and NFs supernatant CCL11concentrations were measured using the CCL11 (human) ELISA kit (R&D system) following the manufacturer’s protocol.

Techniques: Control, Activity Assay, Western Blot, Expressing

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

doi: 10.3390/cancers14133141

Figure Lengend Snippet: CCL11 and CCR3 expression with associated signal pathway in HNC cell lines and their correlation to clinical outcomes in 104 HNC patients. ( a ) Confocal microscopic images showed CCL11 (green) localized to both cell and nuclear membranes, while CCR3 (red) localized only to the cell membrane in FaDu cells; CCL11 and CCR3 co-localized at the cell membrane (yellow). In NPC204 cells, CCL11 (green) and CCR3 (red) were found to co-localize at protrusions polarized to the cells (yellow). ( b ) Using the crisp technique, higher expression of CCR3, MMP2, and MMP3 was found in over-expressed CCL11 cloned-FaDu and NPC204 cells. Cloned CCL11-overexpressed cells were abolished by adding eotaxin siRNA or CCR3 antibody, which reversed the expression of CCR3 and invasion-related MMP2 and MMP9. ( c ) Higher phosphorylation levels of p38 MAPK and ERK were found in cloned CCL11-overexpressed FaDu and NPC204 cells and were reversed by treatment of the p38 MAPK inhibitor (SB203580) and ERK inhibitor (FR180204), respectively. The phosphorylation level of JNK was kept in low condition before and after treatment of the JNK inhibitor (SP600125). ( d ) Photomicrographs of immunohistochemical staining from tissue microarray showing CCL11 and CCR3 expression in three different representative groups of HNC patients (magnification, ×200). ( e ) Kaplan–Meier survival analysis of patients showed that overexpression of CCL11 and CCR3 were statistically associated with poor overall survival).

Article Snippet: CAFs and NFs supernatant CCL11concentrations were measured using the CCL11 (human) ELISA kit (R&D system) following the manufacturer’s protocol.

Techniques: Expressing, Membrane, Clone Assay, Phospho-proteomics, Immunohistochemical staining, Staining, Microarray, Over Expression

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

doi: 10.3390/cancers14133141

Figure Lengend Snippet: The diagrammatic illustration demonstrates the major mechanism that CAFs secreting CCL11 promotes HNC cell migration and invasion and induces properties of drug resistance and stemness, shown as follows. CAFs in TME secret CCL11 binding to the CCR3 receptors on HNC cells via the paracrine effect. The signal induces overexpression of transcriptional factors, such as Snail and Twist, which regulate EMT and are also responsible for self-induction of CCL11 in an autocrine fashion. As a result, CCL11, via paracrine or autocrine signaling when targeting CCR3 receptors, play a functional role in the induction of EMT and CSC properties, for further tumor progression.

Article Snippet: CAFs and NFs supernatant CCL11concentrations were measured using the CCL11 (human) ELISA kit (R&D system) following the manufacturer’s protocol.

Techniques: Migration, Binding Assay, Over Expression, Functional Assay

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Tumor-Associated Dendritic Cells and Colon Cancer Cells Contributes to Tumor Progression via CXCL1

doi: 10.3390/ijms19082427

Figure Lengend Snippet: Upregulation of CXCL1 in DCs isolated from mice and patients with CRC. Increased CXCL1, CXCL2 and CXCL3 ( A ) in SW620-conditioned TADCs, as determined by microarray. Elevated CXCL1 in SW620-conditioned TADCs at mRNA ( B ) and protein ( C ) levels. TADCs were generated by culturing CD14 + monocytes with RPMI, L-15 medium (50%), and SW620-CM (50%) and presenting in GM-CSF (10 ng/mL) and IL-4 (10 ng/mL) for five days. The expressions of mRNA and protein were assessed by microarray, qRT-PCR and Luminex assays. ( D ) The level of CXCL1 in the DCs isolated from patients with CRC. CD11c + cells were isolated from healthy donors and patients with CRC, and the levels of CXCL1 were measured by Luminex assay. ( E ) The levels of CXCL1 in DCs isolated from colon cancer-bearing mice. Mouse colon cancer CT26 cells were injected into mice via intraperitoneal injection. After 14 days, peritoneal lymph nodes were harvested. CD11c + DCs were isolated from the lymph nodes, and the culture medium collected after 24 h incubation. CXCL1 levels were determined by ELISA. Results are representative of at least three independent experiments. Each value is the mean ± SD of three determinations. * Significant difference between the two test groups ( p < 0.05) (*).

Article Snippet: Recombinant human CXCL1 protein was obtained from R&D systems (Minneapolis, MN, USA).

Techniques: Isolation, Microarray, Generated, Quantitative RT-PCR, Luminex, Injection, Incubation, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Tumor-Associated Dendritic Cells and Colon Cancer Cells Contributes to Tumor Progression via CXCL1

doi: 10.3390/ijms19082427

Figure Lengend Snippet: CXCL1 increased cancer stem cell (CSC) properties in SW620 colon cancer cells. Anchorage-independent growth ( A ) and tumor spheroid formation ( B ) of CXCL1-treated SW620 cells. The expression of CD44, CD326 and CD133 ( C ) and aldehyde dehydrogenase (ALDH) activity ( D ) in tumor spheroids. SW620 cells were cultured with CXCL1 protein in soft agar (0.4%) or ultra-low attachment wells for 40 and 10 days, respectively. The tumor spheroids were counted by microscope. ALDH activity and surface markers of tumor spheroids were determined by ALDEFLUOR assay and flow cytometry. ( E ) Level of CSC-related transcription factors in tumor spheroids. Results are representative of at least three independent experiments. Each value is the mean ± SD of three determinations. * Significant difference between the two test groups ( p < 0.05) (*).

Article Snippet: Recombinant human CXCL1 protein was obtained from R&D systems (Minneapolis, MN, USA).

Techniques: Expressing, Activity Assay, Cell Culture, Microscopy, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Tumor-Associated Dendritic Cells and Colon Cancer Cells Contributes to Tumor Progression via CXCL1

doi: 10.3390/ijms19082427

Figure Lengend Snippet: CXCL1 enhanced cancer progression in SW620 cells. CXCL1 increased cell migration, as determined by wound healing ( A ) and transwell ( B ) analysis. The migration ability of SW620 cancer cells was assessed by wound healing assay and transwell assay. CXCL1 acted as a chemoattractant for cancer migration in the transwell system for 24 h. The effect of CXCL1 on the expression of MMP-7 ( C ), EMMPRIN ( D ) and epithelial-to-mesenchymal transition (EMT) ( E ) in SW620 cells. SW620 cells were treated with CXCL1 for 24 h, and the levels of various MMPs and EMT markers were determined by Luminex assay and Western blot. Results are representative of at least three independent experiments. Each value is the mean ± SD of three determinations. * Significant difference between the two test groups ( p < 0.05) (*).

Article Snippet: Recombinant human CXCL1 protein was obtained from R&D systems (Minneapolis, MN, USA).

Techniques: Migration, Wound Healing Assay, Transwell Assay, Expressing, Luminex, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Tumor-Associated Dendritic Cells and Colon Cancer Cells Contributes to Tumor Progression via CXCL1

doi: 10.3390/ijms19082427

Figure Lengend Snippet: Gene Profile of CXCL1-Treated SW620 Cells.

Article Snippet: Recombinant human CXCL1 protein was obtained from R&D systems (Minneapolis, MN, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Tumor-Associated Dendritic Cells and Colon Cancer Cells Contributes to Tumor Progression via CXCL1

doi: 10.3390/ijms19082427

Figure Lengend Snippet: The correlation of CXCL1-regulated genes with CRC patient outcome. The correlation of overall survival ( A ), relapse-free ( B ) and metastasis-free rates ( C ) with CXCL-1 regulated genes. ( D ) The effect of CXCL1 in the expression of PTHLH and TCF4 at mRNA levels. CXCL1 increased the expression of PTHrP ( E ) in SW620 cells. SW620 cells were treated with CXCL1 for 24 h, and the expressions of PTHLH and TCF4 were determined by qRT-PCR and ELISA, respectively. Each value is the mean ± SD of three determinations. * Significant difference between the two test groups ( p < 0.05) (*).

Article Snippet: Recombinant human CXCL1 protein was obtained from R&D systems (Minneapolis, MN, USA).

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Tumor-Associated Dendritic Cells and Colon Cancer Cells Contributes to Tumor Progression via CXCL1

doi: 10.3390/ijms19082427

Figure Lengend Snippet: The effect of CXCL1 on the expression of microRNAs (miRNAs) in colon cancer. ( A ) The regulatory effect of CXCL1 on the expressions of miRNAs in colon cancer. Prognostic significance of miR-105 and miR-597 in overall survival ( B ), relapse-free ( C ) and metastasis-free rates ( D ) in colon cancer patients. The effect of CXCL1 on the levels of miR-105 ( E ) and miR-597 ( F ) in colon cancer. SW620 cells were treated with CXCL1 for 24 h, and the expression of miR-105 and miR-597 determined by qRT-PCR. Each value is the mean ± SD of three determinations. * Significant difference between the two test groups ( p < 0.05) (*). Hsa, Homo sapiens; TCGA, The Cancer Genome Atlas; HR, hazard ratio; PVAL, p -value.

Article Snippet: Recombinant human CXCL1 protein was obtained from R&D systems (Minneapolis, MN, USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Tumor-Associated Dendritic Cells and Colon Cancer Cells Contributes to Tumor Progression via CXCL1

doi: 10.3390/ijms19082427

Figure Lengend Snippet: Proposed model of TADCs in colon cancer. Soluble factors in colon cancer cause abnormal phenotypes of dendritic cells, which in turn increase cancer stem cell properties, cell mobility, and EMT of colon cancer by producing the inflammatory chemokine CXCL1. Transcriptome analysis indicates that CXCL1 increases potential oncogene expression in colon cancer, including PTHLH , TYRP1 , FOXO1 , TCF4 , ZNF880 and the onco-miR miR-105. Our study indicates a new mechanism by which the colon cancer milieu exploits DC plasticity to support cancer progression.

Article Snippet: Recombinant human CXCL1 protein was obtained from R&D systems (Minneapolis, MN, USA).

Techniques: Expressing

Journal: The Journal of pathology

Article Title: Glomerular basement membrane deposition of collagen α1(III) in Alport glomeruli by mesangial filopodia injures podocytes via aberrant signaling through DDR1 and integrin α2β1.

doi: 10.1002/path.5969

Figure Lengend Snippet: Figure 1. Collagen α1(III) is expressed in the glomerular basement membrane in Alport mice. (A) Dual immunofluorescence analysis was per- formed on kidney cryosections from 7-week-old wild-type and Alport mice using antibodies for podocin (a slit diaphragm protein) and DDR1 (a collagen receptor). Clear co-localization is apparent, placing DDR1 at the foot processes (bar = 15 μm). (B) Super-resolution structured illumination microscopy (SR-SIM) of dual immunofluorescence staining of a capillary loop from a 7-week-old Alport mouse stained with anti-DDR1 antibodies (in red) and anti-collagen α1(III) antibodies (in green). The adjacent localization (arrowheads) indicates basement membrane localization of collagen α1(III) (bar = 5 μm). (C) RNA-seq results from wild-type and Alport glomeruli show a marked (>20-fold) increase in the expression of Col3a1 mRNA relative to wild-type. These results were confirmed using real-time RT-PCR (data not shown) and microarray analysis [8]. (D) ImageJ analysis of the relative fluorescence for immunostains of wild-type and Alport glomeruli (six independent glomeruli each) shows significant increases of fluorescence intensity in Alport mice. (E) Western blotting shows clear increases in the 139 kDa band corresponding to collagen α1(III). (F) Quantification of the relative band intensity for triplicate blots of wild-type and Alport mouse glo- meruli indicates significantly elevated abundance of collagen α1(III) in Alport glomeruli relative to wild-type, consistent with the RNA-seq findings. *p < 0.05, ***p < 0.001.

Article Snippet: A DDR1 antibody (AF2396, R&D Systems) was used at 1:75.

Techniques: Membrane, Microscopy, Staining, RNA Sequencing, Expressing, Quantitative RT-PCR, Microarray, Western Blot

Journal: The Journal of pathology

Article Title: Glomerular basement membrane deposition of collagen α1(III) in Alport glomeruli by mesangial filopodia injures podocytes via aberrant signaling through DDR1 and integrin α2β1.

doi: 10.1002/path.5969

Figure Lengend Snippet: Figure 4. Collagen α1(III) activates DDR1 receptors both in vitro and in vivo. (A) Cells were treated or not with collagen III and after 12 h, stained with antibodies against either total DDR1 or phospho-DDR1 (pDDR1) (bar = 5 μm). (B) Cryosections from 7-week-old wild-type and Alport mice were dual immunostained with antibodies specific for pDDR1 or WT1 (a podocyte nuclear marker) (bar = 15 μm). Results indicate that collagen III activates DDR1 receptors both in vitro and in vivo in glomerular podocytes. Arrowheads denote areas of WT1 and pDDR1 co-localization.

Article Snippet: A DDR1 antibody (AF2396, R&D Systems) was used at 1:75.

Techniques: In Vitro, In Vivo, Staining, Marker

Journal: The Journal of pathology

Article Title: Glomerular basement membrane deposition of collagen α1(III) in Alport glomeruli by mesangial filopodia injures podocytes via aberrant signaling through DDR1 and integrin α2β1.

doi: 10.1002/path.5969

Figure Lengend Snippet: Figure 5. The collagen IV α1/α2 network in Alport GBM does not activate DDR1. Cryosections from 5-week-old integrin α1-null Alport mice were stained with antibodies for the indicated proteins. Note the absence of collagen α1(III) in the GBM and the absence of pDDR1 nuclear immunostaining in the podocytes. This indicates that the collagen IV α1/α2 network does not activate DDR1. Bar = 15 μm.

Article Snippet: A DDR1 antibody (AF2396, R&D Systems) was used at 1:75.

Techniques: Staining, Immunostaining

Journal: International Journal of Oncology

Article Title: Genome-wide transcriptional analysis of BRD4-regulated genes and pathways in human glioma U251 cells

doi: 10.3892/ijo.2018.4324

Figure Lengend Snippet: BRD4 knockdown attenuates the proliferation and promotes the apoptosis of U251 cells. (A) A Cell counting kit-8 assay was performed to detect the proliferation of U251 cells transduced with BRD4-shRNA or Scr-shRNA. Each experiment was performed in triplicate. (B) An EdU assay was performed to determine cell proliferation rates following BRD4 knockdown. Nuclei were counterstained blue with DAPI. Red indicates the cells undergoing proliferation. Scale bar, 20 µ m. (C) Quantification of the EdU assay results demonstrated that U251 cell proliferation was reduced following BRD4 knockdown. (D) Representative plots and quantification of flow cytometry analysis of apoptosis following BRD4 knockdown in U251 cells. Data are presented as the mean ± standard deviation of three independent experiments. ** P<0.01. BRD4, bromodomain containing 4; sh, short hairpin; Scr, scrambled control; OD, optical density.

Article Snippet: Scrambled shRNA (Scr-shRNA) that targeted a non-specific sequence (5′-TTCTCCGAACGTGTCACGT-3′) was used as the control. siRNA targeting KRAS proto-oncogene GTPase (KRAS) and the negative control siRNA (cat. nos. sc-35731 and sc-37007, respectively) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Knockdown, Cell Counting, Transduction, shRNA, EdU Assay, Flow Cytometry, Standard Deviation, Control

Journal: International Journal of Oncology

Article Title: Genome-wide transcriptional analysis of BRD4-regulated genes and pathways in human glioma U251 cells

doi: 10.3892/ijo.2018.4324

Figure Lengend Snippet: Ten key genes identified by global signal transduction network analysis.

Article Snippet: Scrambled shRNA (Scr-shRNA) that targeted a non-specific sequence (5′-TTCTCCGAACGTGTCACGT-3′) was used as the control. siRNA targeting KRAS proto-oncogene GTPase (KRAS) and the negative control siRNA (cat. nos. sc-35731 and sc-37007, respectively) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Transduction

Journal: International Journal of Oncology

Article Title: Genome-wide transcriptional analysis of BRD4-regulated genes and pathways in human glioma U251 cells

doi: 10.3892/ijo.2018.4324

Figure Lengend Snippet: Experimental validation of microarray results. (A) Reverse transcription-quantitative polymerase chain reaction results for the mRNA expression levels of the ten key genes identified by global signal transduction network analysis. (B) Western blotting validation of the protein expression changes of key genes in the BRD4-shRNA and the Scr-shRNA groups. GAPDH was used as an internal control. (C) Representative photographs and quantification of KRAS immunostaining in normal brain tissue and glioma tissues of grades II, III and IV. Scale bar, 20 µ m. (D) Western blot analysis of KRAS levels in HA and U251 cells. (E) KRAS silencing following siRNA transfection in U251 cells was confirmed by western blotting (at 72 h post-transfection). (F) A cell counting kit-8 assay was performed to detect the proliferation rates of siKRAS and or siCon-transfected U251 cells. Each experiment was performed in triplicate. (G) The apoptosis rates of siKRAS and siCon-transfected U251 cells were determined by TUNEL staining (red). Nuclei were counterstained with DAPI (blue). Scar bar, 50 µ m. Experimental data are presented as the mean ± standard deviation of at least three experiments. * P<0.05 and ** P<0.01. BRD4, bromodomain containing 4; sh, short hairpin; Scr, scrambled control; KRAS, KRAS proto-oncogene GTPase; HA, human astrocytes; si, small interfering; Con, control; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling; OD, optical density.

Article Snippet: Scrambled shRNA (Scr-shRNA) that targeted a non-specific sequence (5′-TTCTCCGAACGTGTCACGT-3′) was used as the control. siRNA targeting KRAS proto-oncogene GTPase (KRAS) and the negative control siRNA (cat. nos. sc-35731 and sc-37007, respectively) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Biomarker Discovery, Microarray, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Transduction, Western Blot, shRNA, Control, Immunostaining, Transfection, Cell Counting, TUNEL Assay, Staining, Standard Deviation

Journal: Stem Cells Translational Medicine

Article Title: Multiple Autologous Bone Marrow‐Derived CD271 + Mesenchymal Stem Cell Transplantation Overcomes Drug‐Resistant Epilepsy in Children

doi: 10.1002/sctm.17-0041

Figure Lengend Snippet: Molecular and functional analysis of BMMSCs. (A): Percentages of positive cells for subsequent surface markers are given in table. (B): Graphical overview of comparative genomic hybridization microarray results. Expression levels of mRNA for neurotrophic (C) , proangiogenic (D) , and tissue remodeling (E) factors in patients' BMMSCs. qRT‐PCR analysis revealed expression of BDNF, NGF, HGF, PDGF‐A, Flt‐1, VEGF‐A, FGF‐1, FGF‐2, ANGPT‐1, and MMP‐2. Slight expression of GDNF, CNTF, and NGF was observed. No expression of MMP‐9 was detected. The results are mean values ± SD (from triplicates) of mRNA expression relative to expression of housekeeping gene—GAPDH. (F): Western blot detection of VEGF in patients' BMMSCs lysates. Both forms of VEGF were identified: dimer (42 kDa) and monomer (21 kDa). The intermediary band is an effect of nonspecific reaction of antibody, observed also in manufacturer's specification. Standardized protein content in samples from different patients was confirmed by GAPDH detection. (G, H): qRT‐PCR analysis of IDO1 and TSG‐6 mRNA expression levels in patients' BMMSCs stimulated with proinflammatory cytokines. (G): Pretreatment of BMMSCs with INF‐γ caused manifestation of IDO1 expression. (H): Moreover, stimulation of BMMSCs with either TNF‐α or Il‐1β revealed elevated expression of TSG‐6. Pronounced effect was observed for Il‐1β. The results are mean values ± SD (from triplicates) of mRNA expression relative to expression of housekeeping gene—GAPDH. As a control in experiments, BMMSCs in stimulation medium only (2% FBS) were utilized. Abbreviations: ANGPT‐1, angiopoietin 1; BDNF, brain‐derived neurotrophic factor; BMMSC, bone marrow mesenchymal stem cell; CNTF, ciliary neurotrophic factor; FBS, fetal bovine serum; FGF, fibroblast growth factor; Flt‐1, vascular endothelial growth factor receptor 1; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; GDNF, glial‐derived neurotrophic factor; HGF, hepatocyte growth factor; IDO1, indoleamine 2,3‐dioxygenase; Il‐1β, interleukin 1 β; INF‐γ, interferon γ; MMP, metalloproteinase; NGF, nerve growth factor; PDGF, platelet‐derived growth factor; qRT‐PCR, real‐time quantitative reverse transcription polymerase chain reaction; TNF‐α, tumor necrosis factor α; TSG‐6, tumor necrosis factor‐inducible gene 6; VEGF, vascular endothelial growth factor.

Article Snippet: Genes expression were determined by Real‐Time quantitative Reverse Transcription Polymerase Chain Reaction (qRT‐PCR) analysis on ABI PRISM 7300 Sequence Detection System (Applied Biosystems, Foster City, CA, https://www.thermofisher.com/us/en/home/brands/applied-biosystems.html ) using Blank qPCR Master Mix (EURx) and the following Taq‐Man probes (Applied Biosystems) for human genes: glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) ‐ Hs02758991_g1, BDNF ‐ Hs02718934_s1, GDNF ‐ Hs01931883_s1, ciliary neurotrophic factor (CNTF) ‐ Hs04194755_s1, nerve growth factor (NGF) ‐ Hs01113193_m1, HGF ‐ Hs00300159_m1, PDGF‐A ‐ Hs00964426_m1, vascular endothelial growth factor receptor 1 (FLT‐1) ‐ Hs01052937_m1, VEGF‐A ‐ Hs00173626_m1‐, heparin‐binding growth factor 1 (FGF‐1) ‐ Hs01092738_m1, FGF‐2 (Hs00266645_m1), angiopoietin 1 (ANGPT‐1) ‐ Hs00375822_m1, metalloproteinase‐2 (MMP‐2) ‐ Hs00234422_m1, metalloproteinase‐9 (MMP‐9) ‐ Hs00234579_m1, indoleamine 2,3‐dioxygenase (IDO1) ‐ Hs00984148_m1, and tumor necrosis factor‐inducible gene 6 (TSG‐6) ‐ Hs01113602_m1. mRNA expression levels were quantified by the 2 −ΔCt calculation using housekeeping GAPDH as a control.

Techniques: Functional Assay, Hybridization, Microarray, Expressing, Quantitative RT-PCR, Western Blot, Control, Derivative Assay, Reverse Transcription, Polymerase Chain Reaction