Journal: Cancer Management and Research

Article Title:

Cervical Cancer Cells-Secreted Exosomal microRNA-221-3p Promotes Invasion, Migration and Angiogenesis of Microvascular Endothelial Cells in Cervical Cancer by Down-Regulating MAPK10 Expression

doi: 10.2147/cmar.s221527

Figure Lengend Snippet: Figure 1 Low expression of MAPK10 is detected in cervical cancer tissues and cell lines. (A), The heat map of the differentially expressed genes on cervical cancer-related microarray data GSE9750. The abscissa represents the p value, and the ordinate represents the logFC value. Each dot represents a gene, where red refers to the up- regulated gene and green refers to the down-regulated gene in cervical cancer. (B), Enrichment analysis of KEGG metabolic pathways of differentially expressed genes. The abscissa represents the GeneRatio, while the ordinate represents the KEGG item and the upper right histogram represents color gradation. (C), The position and expression of the differentially expressed genes in the TNF signaling pathway. Red refers to highly expressed gene and green refers to poorly expressed gene in cervical cancer. (D), The mRNA expression of MAPK10 in cervical cancer (n = 52) and normal cervical tissues (n = 28) detected by RT-qPCR. (E), The protein expression of MAPK10 in cervical cancer (n = 52) and normal cervical tissues (n = 28) measured by Western blot analysis. (F), Positive rate (× 200) of MAPK10 in cervical cancer (n = 52) and normal cervical tissues (n = 28) determined by IHC assay. *p < 0.05 vs the normal cervical tissues. (G), The mRNA expression of MAPK10 in cervical cancer cell lines (Hela, Caski, SiHa, SW756) and normal cervical epithelial cell line End1/E6E assessed by RT-qPCR. (H), The protein expression of MAPK10 in cervical cancer cell lines (Hela, Caski, SiHa, SW756) and normal cervical epithelial cell line End1/E6E evaluated by Western blot analysis. *p < 0.05 vs the normal cervical epithelial cell line End1/E6E7. The above data are all documented measurement data. Comparisons between two groups were analyzed by non-paired t-test. One-way ANOVA was used for comparing among multiple groups, followed by Tukey’s post hoc test. The experiments were repeated 3 times independently. Abbreviations: KEGG, Kyoto Encyclopedia of Genes and Genomes; TNF, tumor necrosis factor; IHC, Immunohistochemistry; RT-qPCR, reverse transcription quantitative polymerase chain reaction; ANOVA, analysis of variance; MAPK, mitogen-activated protein kinase.

Article Snippet: Cell Culture and Transfection CC cell lines [Caski ((ATCC® CRM-CRL-1550), Hela (ATCC® CCL-2), SiHa (ATCC® HTB-35) and SW756 (ATCC® CRL-10302)] and the normal cervical epithelial cell line End1/E6E7 (ATCC® CRL-2615) [all from American Type Culture Collection (ATCC)] were cultured together in a 37oC incubator supplemented with 5% CO2.

Techniques: Expressing, Microarray, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Cancer Management and Research

Article Title:

Cervical Cancer Cells-Secreted Exosomal microRNA-221-3p Promotes Invasion, Migration and Angiogenesis of Microvascular Endothelial Cells in Cervical Cancer by Down-Regulating MAPK10 Expression

doi: 10.2147/cmar.s221527

Figure Lengend Snippet: Figure 2 Overexpressed MAPK10 leads to inhibited proliferation, migration and invasion of cervical cancer cells. SiHa cells are transfected with oe-MAPK10 and oe-NC. (A), The mRNA expression of MAPK10, the AP-1 family members (c-FOS, c-JUN and JUNB) and angiogenesis related factor VEGF detected by RT-qPCR. (B), The protein expression of MAPK10, the AP-1 family members (c-FOS, c-JUN and JUNB) and angiogenesis related factor VEGF measured by Western blot analysis. (C), Cell viability (× 200) assessed by EdU assay. (D), Cell invasion ability (× 200) determined by Transwell assay. (E), Cell migration ability (× 200) identified by Transwell assay. *p < 0.05 vs SiHa cells transfected with oe-NC. The above data are all documented measurement data. Comparisons between two groups were analyzed by non-paired t-test. The experiments are repeated 3 times independently. Abbreviations: EdU, 5-Ethynyl-2’-deoxyuridine; oe-NC, overexpressed-negative control.

Article Snippet: Cell Culture and Transfection CC cell lines [Caski ((ATCC® CRM-CRL-1550), Hela (ATCC® CCL-2), SiHa (ATCC® HTB-35) and SW756 (ATCC® CRL-10302)] and the normal cervical epithelial cell line End1/E6E7 (ATCC® CRL-2615) [all from American Type Culture Collection (ATCC)] were cultured together in a 37oC incubator supplemented with 5% CO2.

Techniques: Migration, Transfection, Expressing, Quantitative RT-PCR, Western Blot, EdU Assay, Transwell Assay, Negative Control

Journal: Cancer Management and Research

Article Title:

Cervical Cancer Cells-Secreted Exosomal microRNA-221-3p Promotes Invasion, Migration and Angiogenesis of Microvascular Endothelial Cells in Cervical Cancer by Down-Regulating MAPK10 Expression

doi: 10.2147/cmar.s221527

Figure Lengend Snippet: Figure 3 miR-221-3p depletion inhibits cell proliferation, invasion and migration by increasing MAPK10 in cervical cancer. SiHa cells are transfected with miR-221-3p mimic, miR-221-3p inhibitor and oe-MAPK10 + miR-221-3p mimic. (A), Prediction of the upstream regulatory miRNAs of MAPK10. The 4 ellipses represent the predicted results from miRDB database, mirDIP database, TargetScan database and microRNA database, and the middle part represents the intersected results of the 4 databases. (B), Quantitatively analysis on expression of intersected 6 miRNAs in cervical cancer. *p < 0.05 vs the normal cervical tissues. (C), Website Prediction of the binding site between miR-221-3p and MAPK10. (D), The luciferase activity of PGLO-MAPK10 WT and PGLO-MAPK10 MUT in response to the transfection of miR-221-3p mimic detected by dual luciferase reporter gene essay. *p < 0.05 vs the transfection of NC. (E), The expression of miR-221-3p in cervical cancer cell lines (Hela, Caski, SiHa and SW756) and normal cervical epithelial cell line End1/E6E7 tested by RT-qPCR. *p < 0.05 vs normal cervical epithelial cell line End1/E6E7. (F), The mRNA expression of MAPK10, c-FOS, c-JUN, JUNB and VEGF in SiHa cells after transfection detected by RT-qPCR. (G), The protein expression of MAPK10, c-FOS, c-JUN, JUNB and VEGF in SiHa cells after transfection measured by Western blot analysis. (H), Cell viability (× 200) assessed by EdU assay. (I), Cell invasion ability (× 200) determined by Transwell assay. (J), Cell migration ability (× 200) identified by Transwell assay. *p < 0.05 vs SiHa cells transfected with NC mimic. # p < 0.05 vs SiHa cells transfected with NC inhibitor. & p < 0.05 vs SiHa cells transfected with miR-221-3p mimic + oe-NC. The above data are all documented measurement data. Comparisons between two groups were analyzed by non-paired t-test. One-way ANOVA was used for comparison among multiple groups, followed by Tukey’s post hoc test. The experiments are repeated 3 times independently.

Article Snippet: Cell Culture and Transfection CC cell lines [Caski ((ATCC® CRM-CRL-1550), Hela (ATCC® CCL-2), SiHa (ATCC® HTB-35) and SW756 (ATCC® CRL-10302)] and the normal cervical epithelial cell line End1/E6E7 (ATCC® CRL-2615) [all from American Type Culture Collection (ATCC)] were cultured together in a 37oC incubator supplemented with 5% CO2.

Techniques: Migration, Transfection, Expressing, Binding Assay, Luciferase, Activity Assay, Quantitative RT-PCR, Western Blot, EdU Assay, Transwell Assay, Comparison

Journal: Cancer Management and Research

Article Title:

Cervical Cancer Cells-Secreted Exosomal microRNA-221-3p Promotes Invasion, Migration and Angiogenesis of Microvascular Endothelial Cells in Cervical Cancer by Down-Regulating MAPK10 Expression

doi: 10.2147/cmar.s221527

Figure Lengend Snippet: Figure 4 Exosomes derived from cervical cancer cells carry miR-221-3p. (A), Exosome morphology observed by a TEM (scale bar = 200 nm). (B), The expression of exosome surface markers (HSP70, CD9 and CD63) detected by Western blot analysis. (C), The expression of miR-221-3p in exosomes derived from cervical cancer cells measured by RT-qPCR. The above data are all documented measurement data. Comparisons between two groups were analyzed by non-paired t-test. The experiments are repeated 3 times independently *p < 0.05 vs SiHa cells. Abbreviation: TEM, transmission electron microscope.

Article Snippet: Cell Culture and Transfection CC cell lines [Caski ((ATCC® CRM-CRL-1550), Hela (ATCC® CCL-2), SiHa (ATCC® HTB-35) and SW756 (ATCC® CRL-10302)] and the normal cervical epithelial cell line End1/E6E7 (ATCC® CRL-2615) [all from American Type Culture Collection (ATCC)] were cultured together in a 37oC incubator supplemented with 5% CO2.

Techniques: Derivative Assay, Expressing, Western Blot, Quantitative RT-PCR, Transmission Assay, Microscopy

Journal:

Article Title: Pathogenic Natural Antibodies Recognizing Annexin IV Are Required to Develop Intestinal Ischemia-Reperfusion Injury 1

doi: 10.4049/jimmunol.0803980

Figure Lengend Snippet: Characterization of mAb B4 epitope expression. A, Representative flow cytometric histograms show binding of mAb B4 to a single cell suspension of IEC (top), splenocytes (middle) and thymocytes (bottom). Cells were incubated with mAb B4, and bound Ab was detected by using anti-mouse IgM (μ-chain specific) for IEC and thymocytes. In the case of splenocytes, mAb B4 labeled with biotin was used. B and C, Monoclonal Ab B4 epitope expression was determined by Western blot analysis of isolated from thymus, spleen or intestine cell in (B) or lysates were prepared from whole organs in (C). Data are representative of two independent experiments. D, Binding of mAb B4 to proteins typically found as targets of polyreactive natural Abs was studied by micro-array analysis. The positive control for mAb B4 binding are C1q and anti-mouse IgM antibody, positive controls for the micro-array are mAb NC-17D8 and polyclonal IgM, and the negative control is designated blank.

Article Snippet: Polyclonal rabbit anti-annexin IV Ab was obtained from ProteinTech Group, and anti-6xHis mAb was obtained from Novagen.

Techniques: Expressing, Binding Assay, Suspension, Incubation, Labeling, Western Blot, Isolation, Microarray, Positive Control, Negative Control

Journal:

Article Title: Pathogenic Natural Antibodies Recognizing Annexin IV Are Required to Develop Intestinal Ischemia-Reperfusion Injury 1

doi: 10.4049/jimmunol.0803980

Figure Lengend Snippet: Identification of the antigen recognized by mAb B4. A, Lysates prepared from isolated IEC were separated using a sequential three gel separation protocol as described in Materials and Methods. The presence of the B4 antigen was confirmed by Western blot analysis (right). Two gel samples (gray color boxes) were analyzed by MS. B, The MASCOT search results for the protein isolated from first sample was identified as mouse annexin IV based on the highest score number of 785; 78 matched peptides covering 53% of the annexin IV sequence were identified (underlying bold letters). C, Lysates of untransformed F-293 cells (lane 1) and F-293 cells transformed with the pSecTag2/Hygro B expression vector carrying annexin IV insert (F-293-A4) (lane 3) together with culture supernatants from transformed cells (lane 2) were probed by Western blot analysis with mAb B4. D, Flow cytometric analysis of F-293 cells expressing recombinant annexin IV. F-293-A4 cells were probed in flow cytometry with mAb B4. E, Supernatant (lane1) and lysate (lane 2) from F-293-A4 cells expressing recombinant annexin IV that had been incubated in PBS alone, or supernatant (lane 3) and lysate (lane 4) from the same cells incubated with 0.5 M EDTA, were probed by Western blot analysis with mAb B4. Recombinant annexin IV was not released from F-293-A4 cells in the absence of free Ca2+.

Article Snippet: Polyclonal rabbit anti-annexin IV Ab was obtained from ProteinTech Group, and anti-6xHis mAb was obtained from Novagen.

Techniques: Isolation, Western Blot, Sequencing, Transformation Assay, Expressing, Plasmid Preparation, Recombinant, Flow Cytometry, Incubation

Journal:

Article Title: Pathogenic Natural Antibodies Recognizing Annexin IV Are Required to Develop Intestinal Ischemia-Reperfusion Injury 1

doi: 10.4049/jimmunol.0803980

Figure Lengend Snippet: A, Intestinal IR injury is ameliorated in wildtype C57Bl/6 mice receiving recombinant annexin IV prior to the ischemic phase. Reduction of the of IR induced injury to the level of sham operated animals (B6 sham) was observed in wild type mice when they were injected with 50 μg/mouse of annexin IV 5 min prior to the reperfusion phase (A4 IR). Injury in mice pre-injected with control CPK19 protein (CP K19 IR) was comparable with the injury in C57Bl/6 mice undergoing IR (B6 IR) (a). Small intestine tissue samples were processed as in Fig. 6 for myeloperoxidase activity (b) and the eicosanoids LTB4 (c) and PGE2 (d). Each bar is the average ± SEM with 3–6 - animals per group. Statistical significance was determined using one way ANOVA. B, Small intestine of annexin IV pre-injected mice (far right), similarly to sham operated wild type mice (far left) does not show C3 deposition in IR induced injury, in contrast to wild type mice (second from left) and mice pre-injected with the CPK19 control protein undergoing IR (second from right).

Article Snippet: Polyclonal rabbit anti-annexin IV Ab was obtained from ProteinTech Group, and anti-6xHis mAb was obtained from Novagen.

Techniques: Recombinant, Injection, Control, Activity Assay

Journal:

Article Title: Pathogenic Natural Antibodies Recognizing Annexin IV Are Required to Develop Intestinal Ischemia-Reperfusion Injury 1

doi: 10.4049/jimmunol.0803980

Figure Lengend Snippet: Presence of natural antibodies to annexin IV. A, Cr2+/+ in contrast to Cr2−/− mice demonstrate higher levels of IgM natural Ab to annexin IV. Serum samples from three cohorts of mice, Cr2−/− (n=9), Cr2+/+ mice (n=14) and negative control Rag1−/− (n=5) mice were evaluated for the presence of natural IgM (left) and IgG (right) Abs to bacterial annexin IV. The figures are representative of two independent experiments. Statistical significance was determined using a Wilcoxson test. B, 9 serum samples from healthy humans and three from agammaglobulinemic patients were tested in the anti-annexin IV ELISA. Substantial levels of anti-annexin IV IgM antibody IgM is present in the sera of normal humans.

Article Snippet: Polyclonal rabbit anti-annexin IV Ab was obtained from ProteinTech Group, and anti-6xHis mAb was obtained from Novagen.

Techniques: Negative Control, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: Dual inhibition of HDAC and EGFR signaling with CUDC-101 induces potent suppression of tumor growth and metastasis in anaplastic thyroid cancer.

doi: 10.18632/oncotarget.3268

Figure Lengend Snippet: Figure 3: CUDC-101 inhibits ATC cell proliferation, and induces cell cycle arrest and apoptosis. (A) Basal expression of HDAC1, HDAC2 and EGFR in ATC cell lines. (B) Cell proliferation assay. Error bars are mean ± SD. (C) Cell cycle analysis after 24 hours of treatment. (D) The Caspase-Glo 3/7 assay after 48 hours of treatment with CUDC-101. *p < 0.05, **p < 0.01, ***p < 0.001. NS, no significant difference.

Article Snippet: Phospho-kinase and apoptosis protein arrays Human phospho-kinase (Catalog # ARY003B) array (detecting site-specific phosphorylation of 43 kinases and 2 related total proteins, and apoptosis array (Catalog # ARY009 detecting the expression level of 35 apoptosisrelated proteins) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Proliferation Assay, Cell Cycle Assay, Caspase-Glo Assay

Journal: Oncotarget

Article Title: Dual inhibition of HDAC and EGFR signaling with CUDC-101 induces potent suppression of tumor growth and metastasis in anaplastic thyroid cancer.

doi: 10.18632/oncotarget.3268

Figure Lengend Snippet: Figure 5: Protein targets of CUDC-101 in ATC cells. (A) CUDC-101 inhibits HDACs and MAPK in ATC cells. (B) Targets of CUDC-101 identified by phospho-kinase array. Phospho-kinase arrays were performed using cell lysates treated with and without CUDC-101. Shown are those proteins that were altered with CUDC-101 treatment in both ATC cell lines with > 1.5-fold difference. (C) Differentially expressed apoptotic proteins with and without CUDC-101 treatment. Apoptosis arrays were performed. Shown are those proteins that were altered with CUDC-101 treatment in both ATC cell lines with > 1.5-fold difference. For A–C, ATC cells were treated with the vehicle or CUDC-101 at 1.1 μM for 24 hours. (D) CUDC-101 reduces the expression of survivin, XIAP, and β-catenin. Cells were treated with the vehicle or CUDC-101 for 24 hours.

Article Snippet: Phospho-kinase and apoptosis protein arrays Human phospho-kinase (Catalog # ARY003B) array (detecting site-specific phosphorylation of 43 kinases and 2 related total proteins, and apoptosis array (Catalog # ARY009 detecting the expression level of 35 apoptosisrelated proteins) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing