Journal: Molecular Cancer

Article Title: Higher levels of TIMP-1 expression are associated with a poor prognosis in triple-negative breast cancer

doi: 10.1186/s12943-016-0515-5

Figure Lengend Snippet: Analysis of TIMP-1 promoter methylation in breast cancer cell lines and tissues. a Schematic of the TIMP-1 promoter. The black box indicates exon1 and ‘ATG’ indicates the translational start site. Each vertical bar represents a CpG dinucleotide. The CpG island located between −157 to −32 bp includes 11 CpG sites. The arrows indicate the location of the BSP primers. b Methylation status of the TIMP-1 promoter in breast cancer cell lines. BSP analysis was conducted in 10 clones from each cell line. A solid circle represents a methylated CpG site, and an empty circle represents a non-methylated CpG site. The TNBC cell lines (MDA-231 and MDA-468) exhibited lower methylation levels compared with the non-TNBC cell lines (MCF-10A, BT474 and SK-BR-3). c Representative images of the methylation status of TIMP-1 promoter in normal tissue and in various subtypes of breast cancer tissues. d Quantification of the mean methylation levels observed in the cancer tissues. Methylation was reduced in TNBC tissues compared with the benign, luminal-A, luminal-B and HER2+ tissues (left), collectively referred to as the non-TNBC group (right). The data are presented as the mean ± SD, p < 0.05. e Expression of TIMP-1 was analyzed using RT-PCR in breast cancer cells treated with various concentrations (0–20 μM) of 5-Aza for 48 h. TIMP-1 expression was restored in BT474 cells treated with 2–20 μM of 5-Aza. In contrast, TIMP-1 expression was not restored in MDA-231 cells treated with 5-Aza due to hypomethylation of the TIMP-1 promoter

Article Snippet: The human breast cancer cell lines corresponding to the luminal subtype (MCF-7 and BT474 cells), HER2+ subtype (SK-BR-3) and TNBC subtype (MDA-MB-231, MDA-MB-468, MDA-MB-435 and BT549 cells) were obtained from the American Type Culture Collection (ATCC) and cultured according to the manufacturer’s online instructions.

Techniques: Methylation, Clone Assay, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular Cancer

Article Title: Higher levels of TIMP-1 expression are associated with a poor prognosis in triple-negative breast cancer

doi: 10.1186/s12943-016-0515-5

Figure Lengend Snippet: Functional analysis of TNBC cells after TIMP-1 knockdown. a Inactivation of endogenous TIMP-1 in MDA-468 cells using shRNA. The knockdown efficiency was confirmed using real-time PCR and ELISA assays. The data are presented as the mean ± SD. b Inactivation of endogenous TIMP-1 in MDA-231 cells using shRNAs. shTIMP1-1# and shTIMP1-3# were more efficient in knocking down TIMP-1 compared with the sh-control in both cell lines. The data are presented as the mean ± SD. c , d Knockdown TIMP-1 expression induced cell cycle arrest at the G1 phase in MDA-468 and MDA-231 cells. e , f Colony formation was reduced in TIMP-1 knockdown MDA-468 and MDA-231 cells. g CCK-8 assays showed that cell growth was restrained in TIMP-1 knockdown MDA-468 cells. * indicates significant difference as compared to control cells ( p < 0.05). h TIMP-1 knockdown did not significantly disrupt cell invasion in MDA-468 cells. Representative images of invasive cells are shown. The data are presented as the mean ± SD; scale bar, 200 μm

Article Snippet: The human breast cancer cell lines corresponding to the luminal subtype (MCF-7 and BT474 cells), HER2+ subtype (SK-BR-3) and TNBC subtype (MDA-MB-231, MDA-MB-468, MDA-MB-435 and BT549 cells) were obtained from the American Type Culture Collection (ATCC) and cultured according to the manufacturer’s online instructions.

Techniques: Functional Assay, Knockdown, shRNA, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Control, Expressing, CCK-8 Assay

Journal: Molecular Cancer

Article Title: Higher levels of TIMP-1 expression are associated with a poor prognosis in triple-negative breast cancer

doi: 10.1186/s12943-016-0515-5

Figure Lengend Snippet: TIMP-1 regulated cyclin D1 expression by activating the Akt signaling pathway in TNBC cells. a Western blot analyses of cyclin levels in total cell lysates. Cyclin D1 expression decreased in TIMP-1 knockdown MDA-468 cells, whereas cyclin A2, B1 and E2 levels were not significantly affected. b , c RT-PCR and real-time PCR experiments were used to evaluate CCND1 mRNA levels in TIMP-1 knockdown MDA-468 cells. The data are presented as the mean ± SD; #, p < 0.05. d In contrast, cyclin D1 expression was upregulated in MCF-10A cells overexpressing TIMP-1. e Akt (60 kDa) and NF-kB (65 kDa) were inactivated in TIMP-1 knockdown MDA-468 cells, but GSK-3β (46 kDa) and MAPK1/2 (42/44 kDa) activation was not significantly affected. f Exogenous TIMP-1 (100 ng/mL) activated Akt in MDA-468 cells. g Schematic of the potential mechanisms by which TIMP-1 regulates cyclin D1 expression in TNBC cells

Article Snippet: The human breast cancer cell lines corresponding to the luminal subtype (MCF-7 and BT474 cells), HER2+ subtype (SK-BR-3) and TNBC subtype (MDA-MB-231, MDA-MB-468, MDA-MB-435 and BT549 cells) were obtained from the American Type Culture Collection (ATCC) and cultured according to the manufacturer’s online instructions.

Techniques: Expressing, Western Blot, Knockdown, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Activation Assay

Journal: Molecular Cancer

Article Title: Higher levels of TIMP-1 expression are associated with a poor prognosis in triple-negative breast cancer

doi: 10.1186/s12943-016-0515-5

Figure Lengend Snippet: Blocking TIMP-1 activity inhibits TNBC cell growth in vivo. a The effect of the neutralizing antibody against TIMP-1 on TNBC cell growth in vivo. 6-week-old female nude mice were subcutaneously injected with 1 × 10 6 MDA-468 cells. The neutralizing antibody against TIMP-1 or the IgG control was subcutaneously injected into the mice once per week. Representative images of the tumors on week 5 are shown. b Tumor volume (mm 3 ) was evaluated once per week. Tumor growth was significantly inhibited after 4 weeks in mice injected with a neutralizing antibody against TIMP-1 compared with the control mice ( p = 0.0098). The data are presented as the mean ± SD of triplicate measurements. c Tumor weight was measured at the time the mice were sacrificed. Quantification of total tumor burden demonstrated that tumor growth was suppressed by TIMP-1 inhibition. The data are presented as the mean ± SD; n = 13; p = 0.0278

Article Snippet: The human breast cancer cell lines corresponding to the luminal subtype (MCF-7 and BT474 cells), HER2+ subtype (SK-BR-3) and TNBC subtype (MDA-MB-231, MDA-MB-468, MDA-MB-435 and BT549 cells) were obtained from the American Type Culture Collection (ATCC) and cultured according to the manufacturer’s online instructions.

Techniques: Blocking Assay, Activity Assay, In Vivo, Injection, Control, Inhibition

Journal: ACS Omega

Article Title: Novel Fatty Acid in Cordyceps Suppresses Influenza A (H1N1) Virus-Induced Proinflammatory Response Through Regulating Innate Signaling Pathways

doi: 10.1021/acsomega.0c05264

Figure Lengend Snippet: Cytotoxicity and antiviral effect of DDEA in A549 cells and U937 macrophages. For cytotoxicity assay, A549 cells (A) and U937 macrophages (B) were incubated with twofold serial dilution of DDEA for 48 h, followed by MTT staining. The absorbance at a wavelength of 490 nm was measured. In the TCID 50 assay, A549 cells (C) and U937 macrophages (D) were infected by A/Puerto Rico/8/34 (H1N1) (PR8) virus at a multiplicity of infection (MOI) of 1 with indicated concentrations of DDEA. Viral replication in culture supernatants was determined at 24 h postinfection (hpi). Results are presented as the mean ± SD from three independent experiments.

Article Snippet: The U937 cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS, 2.5 mM glutamine, and 10 mM HEPES at 37 °C and differentiated into macrophages-like cells in the presence of 100 nM of phorbol myristate acetate (PMA) (Sigma) for 48 h. Influenza H1N1 virus PR8 strain, A/Puerto Rico/8/34, was purchased from ATCC, propagated in the allantoic cavities of chicken eggs and titrated in MDCK cells.

Techniques: Cytotoxicity Assay, Incubation, Serial Dilution, Staining, Infection, Virus

Journal: ACS Omega

Article Title: Novel Fatty Acid in Cordyceps Suppresses Influenza A (H1N1) Virus-Induced Proinflammatory Response Through Regulating Innate Signaling Pathways

doi: 10.1021/acsomega.0c05264

Figure Lengend Snippet: Inhibitory effect of DDEA on the proinflammatory cytokine expressions in H1N1-infected A549 cells. After PR8 virus (1 MOI) absorption for 2 h, infected cells were treated with or without indicated concentrations of DDEA for 24 h. (A) mRNA expression levels of TNF-α, IFN-α, IFN-β, IL-6, CXCL-8/IL-8, CCL-2/MCP-1, CXCL-10/IP-10, CCL-3/MIP-1α, and CCL-4/MIP-1β were determined by real-time RT–PCR. (B) Levels of proinflammatory cytokines in the culture supernatants were measured by a Bio-Plex assay. Data represented the mean ± SD from three independent experiments. *, p < 0.05, **, p < 0.01, ***, p < 0.001, compared with virus-infected cells without DDEA treatment.

Article Snippet: The U937 cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS, 2.5 mM glutamine, and 10 mM HEPES at 37 °C and differentiated into macrophages-like cells in the presence of 100 nM of phorbol myristate acetate (PMA) (Sigma) for 48 h. Influenza H1N1 virus PR8 strain, A/Puerto Rico/8/34, was purchased from ATCC, propagated in the allantoic cavities of chicken eggs and titrated in MDCK cells.

Techniques: Infection, Virus, Expressing, Quantitative RT-PCR, Plex Assay

Journal: ACS Omega

Article Title: Novel Fatty Acid in Cordyceps Suppresses Influenza A (H1N1) Virus-Induced Proinflammatory Response Through Regulating Innate Signaling Pathways

doi: 10.1021/acsomega.0c05264

Figure Lengend Snippet: Inhibitory effect of DDEA on the proinflammatory cytokine expressions in H1N1-infected U937 macrophages. The PR8 virus at MOI of 1 was first allowed to bind to U937 macrophages for 2 h. After the inoculum was removed, infected cells were treated with or without indicated concentrations of DDEA for another 24 h. (A) mRNA levels of TNF-α, IFN-α, IFN-β, IL-6, CXCL-8/IL-8, CCL-2/MCP-1, CXCL-10/IP-10, CCL-3/MIP-1α, and CCL-4/MIP-1β were detected by real-time RT–PCR. (B) Protein levels of proinflammatory cytokines in the culture supernatant were measured by a Bio-Plex assay. Data represented the mean ± SD from three independent determinations. *, p < 0.05, **, p < 0.01, ***, and p < 0.001, compared with virus-infected cells without DDEA treatment.

Article Snippet: The U937 cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS, 2.5 mM glutamine, and 10 mM HEPES at 37 °C and differentiated into macrophages-like cells in the presence of 100 nM of phorbol myristate acetate (PMA) (Sigma) for 48 h. Influenza H1N1 virus PR8 strain, A/Puerto Rico/8/34, was purchased from ATCC, propagated in the allantoic cavities of chicken eggs and titrated in MDCK cells.

Techniques: Infection, Virus, Quantitative RT-PCR, Plex Assay

Journal: ACS Omega

Article Title: Novel Fatty Acid in Cordyceps Suppresses Influenza A (H1N1) Virus-Induced Proinflammatory Response Through Regulating Innate Signaling Pathways

doi: 10.1021/acsomega.0c05264

Figure Lengend Snippet: DDEA inhibits PRRs- and IFN-mediated signaling pathways activated by H1N1 in A549 cells. The A549 cells were infected by PR8 at MOI of 1 for 2 h. The inoculum was then removed and replaced with medium containing indicated concentrations of DDEA. After 24 h of incubation, total cell lysates were collected and the protein levels of (A) TLR-3, (B) RIG-I, (C) phosphorylated (p)-IRF-3, (D) p-p65, and (E) p-STAT2 were measured by western blot assay. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a housekeeping control.

Article Snippet: The U937 cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS, 2.5 mM glutamine, and 10 mM HEPES at 37 °C and differentiated into macrophages-like cells in the presence of 100 nM of phorbol myristate acetate (PMA) (Sigma) for 48 h. Influenza H1N1 virus PR8 strain, A/Puerto Rico/8/34, was purchased from ATCC, propagated in the allantoic cavities of chicken eggs and titrated in MDCK cells.

Techniques: Protein-Protein interactions, Infection, Incubation, Western Blot, Control

Journal: ACS Omega

Article Title: Novel Fatty Acid in Cordyceps Suppresses Influenza A (H1N1) Virus-Induced Proinflammatory Response Through Regulating Innate Signaling Pathways

doi: 10.1021/acsomega.0c05264

Figure Lengend Snippet: Schematic diagram of mechanisms underlying the anti-inflammatory effect of DDEA on innate immune signaling pathways. Influenza H1N1 virus activates the TLR-3-, RIG-I-, and IFN-dependent signaling pathways. DDEA treatment inhibits a range of molecules in these pathways, including TLR-3, RIG-I, p -IRF-3, p -p65, and p -STAT2.

Article Snippet: The U937 cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS, 2.5 mM glutamine, and 10 mM HEPES at 37 °C and differentiated into macrophages-like cells in the presence of 100 nM of phorbol myristate acetate (PMA) (Sigma) for 48 h. Influenza H1N1 virus PR8 strain, A/Puerto Rico/8/34, was purchased from ATCC, propagated in the allantoic cavities of chicken eggs and titrated in MDCK cells.

Techniques: Protein-Protein interactions, Virus