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: Frontiers in Microbiology

Article Title: Novel Insights Into Cellular Changes in HPV8-E7 Positive Keratinocytes: A Transcriptomic and Proteomic Analysis

doi: 10.3389/fmicb.2021.672201

Figure Lengend Snippet: Transcriptional changes in HPV8-E7 positive keratinocytes. (A) Venn Diagram comparison of two independent cDNA microarray analyses carried out with PHAK expressing HPV8-E7 in the Akgül lab in Cologne and with PHFK expressing HPV8-E7 in the Alonso lab at the DKFZ in Heidelberg. (B) Gene IDs of 4 genes differentially expressed in both array studies. (C,D) GDF15 mRNA expression in isogenic PM1 keratinocytes cultured either in KGM-Gold or RM+, respectively. (E) GDF15 ELISA performed with cell culture supernatants of HPV8-E7 expressing keratinocytes grown in KGM-Gold medium. (F,G) ATF3 mRNA expression in isogenic PM1 keratinocytes cultured either in KGM-Gold or RM+, respectively. (H) GADD34 mRNA expression in PM1 keratinocytes cultured in KGM-Gold. RTqPCRs were normalized to HPRT1 mRNA levels ( n = 3 independent experiments performed in duplicate). Error bars represent standard deviations. Asterisks shown in the figures indicate significant differences of experimental groups in comparison with the corresponding control conditions (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The Human GDF15 Quantikine ELISA Kit (RnD systems) was used to measure GDF15 in cell culture supernatants.

Techniques: Comparison, Microarray, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Control

Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: A. Quantification of change in cell density (number of DAPI positive nuclei per cm 2 imaged area) upon FOXM1 overexpression or knockdown, 72h post transfection. Data is normalized to appropriate controls (Empty vector for pFOXM1 and non-targeting siRNA for siFOXM1). Bars represent Mean + SD (n = 4). P<0.0001 (Student’s t-test). B. Heatmap showing changes in gene expression of a panel of representative markers over a timecourse of RPE culture where cells are seeded at high (100000 cells/cm 2 ) or low (8000 cells/cm 2 ) density. C. Plot showing differential expression of BMP7 and Wnt5B transcripts extrapolated from the microarray data. The shaded area represents 95% confidence intervals around the point estimates (circles) of the difference between the mean high density expression vs the mean low density expression.

Article Snippet: Where required, media was supplemented with recombinant human Wnt5B (500ng/ml; R&D Systems), BMP-4/7 (75ng/ml; R&D Systems), Thiostrepton (Sigma), LDN-193189 (10μM; Stemgent), WAY-262611 (10μM; Enzo Lifesciences).

Techniques: Over Expression, Knockdown, Transfection, Plasmid Preparation, Gene Expression, Quantitative Proteomics, Microarray, Expressing

Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: A. Immunocytochemistry for PMEL17 where cells are seeded at either low density (16000 cells/cm 2 ) in the presence or absence of BMP4/7 (top left) or at high density (25000 cells/cm 2 ) in the presence or absence of Wnt5B (bottom left) and cultured for a period of 14 days. Also shown is the expression of BEST1 under the same conditions (top and bottom right). ACTB and B2M are used as housekeeping genes. Bars represent Mean + SD (n = 3). B. Quantification of immunocytochemistry for % CRALBP at Day 21 where cells are either treated with media alone (Control) or media supplemented with 10μM LDN-193189 added at Day 2,4,6,8,11,14 or 18. * indicates significant difference between control and compound treatment (One way ANOVA with Dunnett’s multiple comparisons). C. Quantification of immunocytochemistry for % CRALBP at Day 28 where cells are either treated with media alone (Control) or media supplemented with 10μM WAY-262611 added at Day 2,7,14 or 21. * indicates significant difference between control and compound treatment (One way ANOVA with Dunnett’s multiple comparisons). D. qPCR based measurement of BMP7 and Wnt5B transcript expression at Day 10 post siFOXM1 transfection (relative to transfection with non-targeting siRNA used as a control). GAPDH , HPRT1 and IPO8 were used as housekeeping genes. Bars represent Mean + SD (n = 3). P<0.05 (Student’s t-test).

Article Snippet: Where required, media was supplemented with recombinant human Wnt5B (500ng/ml; R&D Systems), BMP-4/7 (75ng/ml; R&D Systems), Thiostrepton (Sigma), LDN-193189 (10μM; Stemgent), WAY-262611 (10μM; Enzo Lifesciences).

Techniques: Immunocytochemistry, Cell Culture, Expressing, Control, Transfection

Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: RPE first acquire a mesenchymal morphology upon dissociation and culture followed by proliferation and mesenchymal-epithelial transition to re-uptake an epithelial phenotype. Proliferation of RPE is directly regulated by FOXM1 which also affects expression of BMP7 and Wnt5B by an unknown mechanism. Both these activities are required for successful MET and epithelialization.

Article Snippet: Where required, media was supplemented with recombinant human Wnt5B (500ng/ml; R&D Systems), BMP-4/7 (75ng/ml; R&D Systems), Thiostrepton (Sigma), LDN-193189 (10μM; Stemgent), WAY-262611 (10μM; Enzo Lifesciences).

Techniques: Expressing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Immunohistochemistry staining with antibody against phosphorylated Smad2 (pSmad2) and TGFβ receptor II (TβRII) showed increased nuclear signal for pSmad2 in the invasive ECdnT organotypic cultures. Scale bar 50 micron. (b) Analysis of immunohistochemistry staining for TβRII and pSmad2 in 83 ESCC cases in a tissue microarray shows no significant correlation. Fisher’s exact test, two tailed p= 0.3182. (c) Five paired normal adjacent and ESCC tissues (GSE17531) were analyzed for INHBA mRNA expression, which identified upregulation of INHBA in four ESCC samples. (d) Waterfall plot of a publically available dataset (GSE23400) represented upregulation of INHBA in the ESCC (grey bars) samples vs. normal (black bars).

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Immunohistochemistry, Staining, Microarray, Two Tailed Test, Expressing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Esophageal epithelial cells expressing wild-type full-length E-cadherin (E), dominant-negative mutant E-cadherin (EC) or dominant-negative mutant E-cadherin and TGFβ receptor II (ECdnT) were grown in organotypic cultures with either fetal esophageal fibroblasts (FEF) or cancer-associated fibroblasts (CAF) embedded in the underlying matrix. Immunofluorescence staining with antibody against αSMA (green) and podoplanin (red) showed similar expression pattern in the cultures. Scale bar is 50 micron. (b) Activin A concentration in conditioned media from organotypic cultures is higher in invasive cultures as measured using indirect ELISA. * p=0.003, ** p= 0.005, *** p=0.03 (c) Stimulation of epithelial cells with Act A in monolayer plastic culture demonstrated phosphorylation of Smad. Neutralizing antibody against Activin (nAb) prevented the induction of pSmad2 by Act A. Following stimulation with Act A or with conditioned media from organotypic culture increased expression of vimentin was detected after 48 hours by Western Blot. The increase was reversed in the presence of neutralizing antibody (nAb). (d) Inhibition with the Act A antagonist, Follistatin, or a pan-TGFβ inhibitor A83-01 suppressed MMP-9 secretion in E, EC and ECdnT cells as measured by gelatin zymography. Upper bands reflect pro-MMP, lower bands activated, cleaved MMP (arrow).

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Expressing, Dominant Negative Mutation, Immunofluorescence, Staining, Concentration Assay, Indirect ELISA, Phospho-proteomics, Western Blot, Inhibition, Zymography

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Esophageal epithelial cells expressing wild-type full-length E-cadherin (E), dominant-negative mutant E-cadherin (EC) or dominant-negative mutant E-cadherin and TGFβ receptor II (ECdnT) were grown in organotypic cultures in the presence of recombinant Activin A (Act A), its antagonist Follistatin or a neutralizing antibody against Activin A (nAb); H&E staining. Stimulation with Act A inhibited invasion of E and EC cells, but failed to suppress ECdnT cell invasion. Follistatin increased cell invasion in all cell types, while the neutralizing antibody prevented invasion of E and EC cells, without an effect on ECdnT cells. (b) Immunohistochemistry staining with ki67-antibody showed no differences in cell proliferation. Scale bars are 50 micron. (c) Indirect ELISA with antibody against Act A measured increased levels after addition of recombinant Act A in fibroblasts (FEF) and ECdnT. Untreated ECdnT cells (Control) secreted higher baseline levels of Act A than FEF, which were reduced by Follistatin. (d) TGFβ1 concentration was increased in response to stimulation with Act A and overall baseline secretion was higher in control ECdnT cells than fibroblasts as measured by indirect ELISA. Follistatin inhibited TGFβ1 secretion.

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Expressing, Dominant Negative Mutation, Recombinant, Staining, Immunohistochemistry, Indirect ELISA, Control, Concentration Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Separating the cellular matrix and epithelium of the organotypic cultures growing ECdnT cells through a collagen I layer, dashed lines, prevented cell invasion in the absence (control) and presence of Act A (+Act A). When the cellular matrix of the organotypic culture was treated with puromycin to kill the embedded fibroblasts before the ECdnT cells were seeded, epithelial formation occurred but invasion was inhibited with and without Act A stimulation. (b) Treatment of ECdnT organotypic cultures with a pan-MMP inhibitor, GM6001, suppressed cell invasion, which was not restored in the presence of Act A. Untreated (no tx) control ECdnT cells in organotypic culture invaded into the underlying matrix. TGFβ1 treatment inhibited epithelial cell invasion. Scale bars are 50 micron. (c) Immunohistochemistry showed nuclear localization of phosphorylated Smad (pSmad2, red) in control and Act A stimulated conditions. Collagen IV, red, was disrupted in invasive cultures after Act A treatment. Loss of the fibroblasts (FEF), labeled green with antibody against vimentin (no staining in the lower panels), had no effect on the nuclear localization of pSmad2. The collagen IV layer was not disrupted in non-invasive cultures in the absence of FEFs.

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Control, Immunohistochemistry, Labeling, Staining

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: Immunity

Article Title: Stellate Cells, Hepatocytes, and Endothelial Cells Imprint the Kupffer Cell Identity on Monocytes Colonizing the Liver Macrophage Niche

doi: 10.1016/j.immuni.2019.08.017

Figure Lengend Snippet:

Article Snippet: Recombinant Human/Mouse/Rat BMP-2 Protein , RnD Systems , Cat#355-BM-010.

Techniques: Control, Recombinant, Blocking Assay, Irradiation, Enzyme-linked Immunosorbent Assay, cDNA Synthesis, Microarray, Software, Microscopy

Journal: Frontiers in Immunology

Article Title: RNA-Seq Analysis of IL-1B and IL-36 Responses in Epidermal Keratinocytes Identifies a Shared MyD88-Dependent Gene Signature

doi: 10.3389/fimmu.2018.00080

Figure Lengend Snippet: Type I and II interferon genes have time-dependent IL-1B and IL-36 responses. (A) Interferon gamma receptor 1 ( IFNGR1 ). (B) Interferon gamma receptor 2 ( IFNGR2 ). (C) Interferon alpha and beta receptor subunit 2 ( IFNAR2 ). In panels (A–C) , average FPKM (±1 SE) is shown for each group, and asterisks denote significant differences relative to the control (CTL) treatment at the corresponding time point (paired two-sample t -test; n = 2 or 3 per treatment). (D) Genes with interferon response factor 1 (IRF1) binding sites (5 kb upstream region). The middle 50% of FC estimates is shown for genes with 2+ IRF1 binding sites compared with genes with fewer IRF1 sites (magenta font, horizontal axis: P < 0.05, Wilcoxon rank sum test). The IRF1 position weight matrix is shown (right) along with the IRF1 tetrameric structure (bottom right; NCBI structure database). (E) IFN-induced gene signature scores. IFN-induced genes were identified from microarray studies of IFN-treated keratinocytes (left margin), and the average FC for these genes was calculated in IL-1B/IL-36 experiments (bottom margin). Left margin labels indicate the cytokine concentration (in ml), treatment duration, and GEO series accession number. All cytokine experiments were replicated with at least two samples per treatment. (F) Top 30 IFN-g-induced genes (identified from GSE36287). (G) Top 35 INFa-induced genes (identified from GSE36287). (H) Self-organizing maps (SOMs). The SOM layout was determined only from IFN-g-induced genes (i.e., 2,500 genes most strongly induced by IFN-g, GSE36287). Colors reflect average FC estimates for IFN-g-induced genes assigned to each SOM region (columns 1 and 2 on left). The final column (yellow–blue) displays the mean FC difference for each cytokine with respect to each SOM region (8 h mean FC–24 h mean FC; log 2 scale).

Article Snippet: MYD88-KO KCs including WT KCs were grown in 12-well plates, and cells were treated with recombinant IL-1 beta (10 μg/ml; R&D Systems # 201-LB-025), IL-36 gamma (10 μg/ml; R&D Systems # 6835-IL-010), IFN-gamma (50 μg/ml; R&D Systems # 285-IF-100), IL-17A (20 μg/ml; R&D Systems # 317-ILB-050), and/or TNF-alpha (10 μg/ml; R&D Systems # 210-TA-005) for 8 or 24 h. RNAs were isolated from cell cultures using Qiagen RNeasy plus kit (Cat # 74136).

Techniques: Control, Binding Assay, Microarray, Concentration Assay

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: Development (Cambridge, England)

Article Title: Severe pre-eclampsia is associated with alterations in cytotrophoblasts of the smooth chorion

doi: 10.1242/dev.146100

Figure Lengend Snippet: Confirmation of the microarray results: protein level and functional data. CSH1, GSTA3 and PAPPA1 immunoreactivity corroborated differential expression at the RNA level. The identity of cytotrophoblasts in the smooth chorion (schCTBs) was confirmed by cytokeratin (CK) expression. (A) Little to no signal for CSH1 was detected in cases of non-infected preterm birth (nPTB). In contrast, a subset of schCTBs interspersed among the immunonegative cells stained strongly with an antibody that recognized this molecule. (B) In sPE, the same pattern of differential expression was observed for GSTA3 except that immunoreactivity was more widespread among the schCTBs and the signal was associated with cells that were adjacent to the decidua parietalis. (C) Immunolocalization of PAPPA in the fetal membranes showed high CTB-associated immunoreactivity in sPE compared with largely background staining in nPTB. The images are representative of the analysis of a minimum of three sections from different areas of smooth chorion biopsies for each case ( n =4/group). Cytokeratin (CK7) expression confirmed trophoblast identity and DAPI staining enabled visualization of nuclei. a, amnion; dec, decidua. Scale bars: 100 μm. (D) ELISA quantification of CTB PAPPA1 secretion into the culture medium. In sPE, vCTB and schCTB release of PAPPA1 significantly increased compared with the same subpopulations of cells in nPTB. n =3/group. (E) Isolated schCTBs and vCTBs ( n =3 second trimester samples/group) were cultured in the presence of exogenous IGF1. Compared with baseline (no addition), IGF1 (2 and 10 ng) increased BrDU incorporation by schCTB, but not vCTBs. * P <0.05; ** P <0.01.

Article Snippet: Freshly isolated second trimester vCTBs and schCTBs were plated at a density of 8400 cells per well of a 96-well plate coated with Matrigel diluted 1:2 in medium containing various concentrations of human recombinant IGF1 (291-G1-200, R&D Systems): 0, 2, 10, 50 and 75 ng/ml.

Techniques: Microarray, Functional Assay, Quantitative Proteomics, Expressing, Infection, Staining, Enzyme-linked Immunosorbent Assay, Isolation, Cell Culture, BrdU Incorporation Assay

Journal:

Article Title: Enhanced Expression of Keratinocyte Growth Factor and Its Receptor Correlates with Venous Invasion in Pancreatic Cancer

doi: 10.2353/ajpath.2007.060935

Figure Lengend Snippet: Effect of KGF on VEGF-A expression in pancreatic cancer cells. A: Time course of VEGF-A induction. Q-PCR analysis showed significant increases in VEGF-A mRNA levels at 3 and 6 hours after the addition of 10 ng/ml rhKGF to MIA PaCa-2 cells. Each experiment was performed twice, and gene expression measurements were performed in triplicate. Bars represent the mean ± SE (*P = 0.038, **P = 0.041). B: Effects of exogenous KGF on VEGF-A protein levels. After the addition of rhKGF (0 to 100 ng/ml) to MIA PaCa-2 cells, VEGF-A levels in the culture supernatant were measured by ELISA and were found to be increased significantly in a dose-dependent manner. Results shown represent the mean ± SE of two separate experiments, each conducted in triplicate (*P = 0.034, **P = 0.018). C and D: Effects of engineered expression of KGF on VEGF-A expression. PANC-1-KGF and PANC-1-Mock cells were incubated in serum-free medium for 72 hours. KGF and VEGF-A mRNA levels were then determined by Q-PCR analysis. KGF (*P < 0.0001; C) and VEGF-A (*P = 0.004; D) mRNA levels in PANC-1-KGF were significantly higher than in PANC-1-Mock cells. Each experiment was performed twice, and each measurement was performed in triplicate. Bars represent the mean ± SE. E and F: Effects of sh-KGFR on VEGF-A expression. Western blot analysis showed that sh-KGFR inhibited the expression of KGFR protein levels in MIA PaCa-2 cells compared with sh-control (*P = 0.0006; E). Cells were treated with rhKGF (10 ng/ml) after being transfected with sh-KGFR or sh-control. sh-KGFR transfection was associated with significantly reduced VEGF-A protein levels in the conditioned medium after the addition of rhKGF (10 ng/ml) compared with sh-control (*P = 0.0016; F). Each measurement was performed in triplicate. Bars represent the mean ± SE. G and H: Effects of sh-KGF on VEGF-A expression. sh-KGF and sh-control were transiently transfected into KLM-1 cells. Q-PCR revealed that sh-KGF significantly reduced KGF and VEGF-A mRNA levels in KLM-1 cells compared with sh-control (*P = 0.0002, **P = 0.0046; G). Furthermore, sh-KGF significantly reduced VEGF-A protein levels in conditioned medium compared with sh-control (*P = 0.0016; H). Each experiment was performed twice, and each measurement was performed in triplicate. Bars represent the mean ± SE.

Article Snippet: The chemicals and reagents were purchased as follows: Isogen from Nippon Gene (Tokyo, Japan); a Takara RNA PCR kit (AMV) version 3.0 and pBAsi-hU6 Neo DNA vector from Takara Biotech (Tokyo, Japan); RNeasy mini kit from Qiagen GmbH (Hilden, Germany); Transcriptor First Strand cDNA Synthesis kit and LightCycler FastStart DNA Master SYBR Green I, FuGENE 6, and FuGENE HD transfection reagent from Roche Diagnostics GmbH (Mannheim, Germany); human VEGF Quantikine Colorimetric Sandwich enzyme-linked immunosorbent assay (ELISA) kit, goat polyclonal anti-FGF-7 antibodies, and recombinant human KGF (rhKGF) from R&D Systems Inc. (Westerville, OH); Immobilon P transfer membrane from Millipore (Yonezawa, Japan); M-PER Mammalian Protein Extraction reagent and Super Signal West Pico chemiluminescent substrates from Pierce (Rockford, IL); SERVA Blau G from Serva Electrophoresis GmbH (Heidelberg, Germany); Histofine Simple Stain Max PO (G) or (R) kit from Nichirei Biosciences, Inc. (Tokyo, Japan); anti-rabbit IgG-horseradish peroxidase secondary antibody and rabbit polyclonal anti-VEGF-A antibodies (A-20) from Santa Cruz Biotechnology (Santa Cruz, CA); Human Tissue Microarray 1 and Human Digestive Tissue Sets from Novagen (Darmstadt, Germany); fluorescein 5-isothiocyanate-conjugated anti-rabbit IgG and Vectashield mounting medium containing 4′,6-diamidino-2-phenylindole dihydrochloride from Vector Laboratories, Inc. (Burlingame, CA); silane-coated slides and a malinol mounting medium from Muto Pure Chemicals Co., Ltd. (Tokyo, Japan); and pIRES2-EGFP vector from Clontech (Palo Alto, CA).

Techniques: Expressing, Gene Expression, Enzyme-linked Immunosorbent Assay, Incubation, Western Blot, Control, Transfection

Journal: Cancer Research

Article Title: The BH3 Mimetic ABT-737 Induces Cancer Cell Senescence

doi: 10.1158/0008-5472.can-10-1977

Figure Lengend Snippet: Figure 1. Gene transcription changes after ABT-737 treatment. A, microarray analysis of gene expression induced by ABT-737. PV-10 cells were treated in triplicate with DMSO or 10 mmol/L ABT-737 for 24 hours and gene microarray changes documented. B, gene changes associated with senescence. C, qT-PCR analysis of IL-6 and IL-8 transcripts. PV-10 and 22Rv1 cells were treated in triplicate with DMSO, ABT-737 (10 mmol/L) or enantiomer (En., 10 mmol/ L) for 24 hours (mean SD, n ¼ 3). D, the secretion of IL-6 and IL-8 in PV-10 cells treated with DMSO, ABT-737 or enantiomer for 24 hours was determined by ELISA (n ¼ 6, mean SD).

Article Snippet: Conditioned medium was collected following treatment and used at a 2:1 dilution in the human interleukin-6 (IL-6) or IL-8 ELISA Kit (R&D Systems) according to the manufacturer's instructions.

Techniques: Microarray, Gene Expression, Enzyme-linked Immunosorbent Assay