Journal: Frontiers in Microbiology

Article Title: Relationship between Milk Microbiota, Bacterial Load, Macronutrients, and Human Cells during Lactation

doi: 10.3389/fmicb.2016.00492

Figure Lengend Snippet: Relationship between number of somatic cells and bacterial load in milk samples . The graph shows a comparison between the number of bacterial cells per milliliter (estimated by qPCR) and the number of somatic cells per milliliter, estimated with an Integrated Milk Testing Fossomatic 5000 (FOSS) cytometer. ( n = 38, R 2 = 0.0066). C, colostrum samples ( n = 12); T, transition samples ( n = 15); M, mature samples ( n = 11).

Article Snippet: Each reaction mixture of 20 μl was composed of KAPA Sybr Fast qPCR Kit (KAPA Biosistems), 0.4 μl of each primer (10 μM concentration) and 1 μl of template DNA using an annealing temperature of 62°C in a Light Cycler 480 Real-Time PCR System (Roche Technologies).

Techniques: Comparison, Cytometry

Journal: Frontiers in Microbiology

Article Title: Relationship between Milk Microbiota, Bacterial Load, Macronutrients, and Human Cells during Lactation

doi: 10.3389/fmicb.2016.00492

Figure Lengend Snippet: Richness and diversity of milk samples. (A) Shows the richness in the samples as inferred by computation of Chao1 index, compared with bacterial load in cells per ml, as estimated by qPCR. (B) Shows the diversity in the samples as inferred by Shannon index, compared with bacterial load. ( n = 30 in both cases).

Article Snippet: Each reaction mixture of 20 μl was composed of KAPA Sybr Fast qPCR Kit (KAPA Biosistems), 0.4 μl of each primer (10 μM concentration) and 1 μl of template DNA using an annealing temperature of 62°C in a Light Cycler 480 Real-Time PCR System (Roche Technologies).

Techniques:

Journal: The Journal of Veterinary Medical Science

Article Title: Induction of pluripotency in large Japanese field mouse ( Apodemus speciosus ) fibroblasts via reprogramming

doi: 10.1292/jvms.25-0390

Figure Lengend Snippet: Quantitative PCR (qPCR) analysis of embryoid bodies (EBs) derived from Apodemus speciosus induced pluripotent stem cells (iPSCs) at passage 13 cultured in the 2i condition. Relative expression levels of Tubb3 , Nestin , T , Acta2 , Gata4 , and Afp . “fib”, “iPSC”, “EBd7” and “EBd14” refer to fibroblasts, iPSCs, EBs cultured during 7 days and EBs cultured during 14 days, respectively. Gapdh was used as endogenous control and the expression level in Apodemus speciosus iPSCs was set to 1.0. * indicates P <0.05.

Article Snippet: qPCR was conducted using the KAPA SYBR Fast qPCR Kit (Nippon Genetics) with synthesized cDNA as the template.

Techniques: Real-time Polymerase Chain Reaction, Derivative Assay, Cell Culture, Expressing, Control

Journal: PLoS ONE

Article Title: Decrease of miR-146b-5p in Monocytes during Obesity Is Associated with Loss of the Anti-Inflammatory but Not Insulin Signaling Action of Adiponectin

doi: 10.1371/journal.pone.0032794

Figure Lengend Snippet: Activation of monocytes in the circulation is characterized by increased activation of the inflammatory toll-like receptor-2 (TLR2)/NFκB signaling pathway. Activation of this pathway will elicit pro-inflammatory cytokine release (e.g. tumor necrosis factor α (TNFα)), mitochondrial and intracellular ROS production and impairment of the insulin sensitivity at the molecular level. More mitochondrial ROS production is associated with an increased expression of the mitochondrial antioxidant superoxide dismutase-2 ( SOD2 ), suggesting that SOD2 only acts as mitochondrial stress marker (indicated as dotted line). The formed ROS will not only induce the IKK complex phosphorylation and thereby the translocation of NFκB but will also lead to inhibition of the insulin receptor signaling pathway. Interestingly, interleukin-1 receptor-associated kinase-3 (IRAK3) and miR-146b-5p are located upstream of the transcription factor NFκB and negatively regulates TLR signaling. IRAK3 prevents the dissociation of IRAK1 and IRAK4 from MyD88 and formation of IRAK-TRAF6 complexes. miR-146b-5p, on the other hand, inhibits the translation of IRAK1 and TRAF6. Flow of the pathway at the protein interaction level is indicated by black arrows. Blunted arrows indicate inhibition. Phosphorylation is indicated by a circled P.

Article Snippet: NFκB p65 DNA binding activity was assessed on isolated nuclear extracts of transfected THP-1 monocytes by ELISA using the TransAM NFκB p65 transcription factor assay kit according to the manufacturer's protocol (Active Motif, La Hulpe, Belgium).

Techniques: Activation Assay, Expressing, Marker, Phospho-proteomics, Translocation Assay, Inhibition

Journal: PLoS ONE

Article Title: Decrease of miR-146b-5p in Monocytes during Obesity Is Associated with Loss of the Anti-Inflammatory but Not Insulin Signaling Action of Adiponectin

doi: 10.1371/journal.pone.0032794

Figure Lengend Snippet: ( A ) IRAK1 and TRAF6 protein levels, ( B ) NFκB p65 DNA binding activity and TNFα RNA levels, ( C ) IRAK3 RNA and protein levels, ( D ) m- and iROS production and ( E ) IRS1 RNA and protein levels in THP-1 cells transfected with miR-146b-5p inhibitor as determined by qRT-PCR, ELISA, Western blotting and flow cytometry. Data are expressed as means ± SEM. n = 6. * P <0.05 and ** P <0.01 compared with THP-1 cells transfected with negative control miRNA. Abbreviations: iROS, intracellular ROS; mROS, mitochondrial ROS; ROS, reactive oxygen species.

Article Snippet: NFκB p65 DNA binding activity was assessed on isolated nuclear extracts of transfected THP-1 monocytes by ELISA using the TransAM NFκB p65 transcription factor assay kit according to the manufacturer's protocol (Active Motif, La Hulpe, Belgium).

Techniques: Binding Assay, Activity Assay, Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Flow Cytometry, Negative Control

Journal: Molecular Oncology

Article Title: Long noncoding RNA LINC01578 drives colon cancer metastasis through a positive feedback loop with the NF‐κB/YY1 axis

doi: 10.1002/1878-0261.12819

Figure Lengend Snippet: LINC01578 is upregulated by NF‐κB and YY1. (A) The predicted NF‐κB and YY1 binding sites on LINC01578 promoter. NF‐κB and YY1 bind sites were at −438 and −4 positions relative to the transcription start site, respectively. (B) ChIP assays were performed in DLD‐1 cells to measure the binding of NF‐κB and YY1 on LINC01578 promoter. A distant region without NF‐κB and YY1 binding sites was used as NC (P3). (C) Luciferase reporter assays for DLD‐1 cells transfected with luciferase reporter plasmids containing LINC01578 promoter and treated with PBS or 10 ng·mL −1 TNF‐α for 24 h. Nuclear p65 levels of DLD‐1 cell treatment with PBS or 10 ng·mL −1 TNF‐α for 24 h were detected by western blot. (D) Luciferase reporter assays for DLD‐1 cells transfected with luciferase reporter plasmids containing LINC01578 promoter and treated with DMSO or 5 µ m JSH‐23 for 24 h. Nuclear p65 levels of DLD‐1 cell treatment with DMSO or 5 µ m JSH‐23 for 24 h were detected by western blot. (E) Luciferase reporter assays for DLD‐1 cells cotransfected with luciferase reporter plasmids containing LINC01578 promoter and p65 overexpression vector. p65 overexpression efficiencies were detected by western blot. (F) Luciferase reporter assays for DLD‐1 cells cotransfected with luciferase reporter plasmids containing LINC01578 promoter and siRNAs against p65. p65 knockdown efficiencies were detected by western blot. (G) Luciferase reporter assays for DLD‐1 cells cotransfected with luciferase reporter plasmids containing LINC01578 promoter and YY1 overexpression vector. YY1 overexpression efficiencies were detected by western blot. (H) Luciferase reporter assays for DLD‐1 cells cotransfected with luciferase reporter plasmids containing LINC01578 promoter and siRNAs against YY1. YY1 knockdown efficiencies were detected by western blot. (I) LINC01578 expression in DLD‐1 cells treated with PBS or 10 ng·mL −1 TNF‐α for 24 h. (J) LINC01578 expression in DLD‐1 cells treated with DMSO or 5 µ m JSH‐23 for 24 h. (K) LINC01578 expression in DLD‐1 cells transfected with p65 overexpression vector. (L) LINC01578 expression in DLD‐1 cells transfected with siRNAs against p65. (M) LINC01578 expression in DLD‐1 cells transfected with YY1 overexpression vector. (N) LINC01578 expression in DLD‐1 cells transfected with siRNAs against YY1. Data are shown as mean ± SD based on three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, not significant, by one‐way ANOVA followed by Dunnett's multiple comparisons test (B) or Student's t ‐test (C‐N).

Article Snippet: The nuclear extracts from indicated cells were used to measure NF‐κB transcription factor DNA binding activities using the NF‐κB p50 Transcription Factor Assay Kit (Cat. ab207217; Abcam) and NF‐κB p65 Transcription Factor Assay Kit (Cat. ab133112; Abcam) following the manufacturer's protocols.

Techniques: Binding Assay, Luciferase, Transfection, Western Blot, Over Expression, Plasmid Preparation, Expressing

Journal: Molecular Oncology

Article Title: Long noncoding RNA LINC01578 drives colon cancer metastasis through a positive feedback loop with the NF‐κB/YY1 axis

doi: 10.1002/1878-0261.12819

Figure Lengend Snippet: LINC01578 activates NF‐κB signaling. (A) p50 IHC staining of liver metastatic tumors derived from LINC01578‐overexpressed and control DLD‐1 cells. Scale bars, 50 µm. (B) p50 IHC staining of liver metastatic tumors derived from LINC01578‐depleted and control LoVo cells. Scale bars, 50 µm. (C) Nuclear p50 and p65 levels in LINC01578‐overexpressed and control DLD‐1 cells were detected by western blot. (D) Nuclear p50 and p65 levels in LINC01578‐depleted and control LoVo cells were detected by western blot. (E) Firefly luciferase reporters containing NF‐κB binding sites (pNFκB‐luc) were transfected into LINC01578‐overexpressed and control DLD‐1 cells. Then, luciferase reporter assays were performed to determine NF‐κB transcriptional activity. (F) Firefly luciferase reporters containing NF‐κB binding sites (pNFκB‐luc) were transfected into LINC01578‐depleted and control LoVo cells. Then, luciferase reporter assays were performed to determine NF‐κB transcriptional activity. (G) p50 and p65 activation in nuclear extracts from LINC01578‐overexpressed and control DLD‐1 cells was determined by NF‐κB p50 Transcription Factor Assay Kit and NF‐κB p65 Transcription Factor Assay Kit, respectively. (H) p50 and p65 activation in nuclear extracts from LINC01578‐depleted and control LoVo cells was determined by NF‐κB p50 Transcription Factor Assay Kit and NF‐κB p65 Transcription Factor Assay Kit, respectively. Data are shown as mean ± SD based on n = 6 mice in each group (A,B) or three independent experiments (C–H). * P < 0.05, ** P < 0.01 by the Mann–Whitney test (A), Kruskal–Wallis test followed by Dunn's multiple comparisons test (B), or one‐way ANOVA followed by Dunnett's multiple comparisons test (E–H).

Article Snippet: The nuclear extracts from indicated cells were used to measure NF‐κB transcription factor DNA binding activities using the NF‐κB p50 Transcription Factor Assay Kit (Cat. ab207217; Abcam) and NF‐κB p65 Transcription Factor Assay Kit (Cat. ab133112; Abcam) following the manufacturer's protocols.

Techniques: Immunohistochemistry, Derivative Assay, Western Blot, Luciferase, Binding Assay, Transfection, Activity Assay, Activation Assay, Transcription Factor Assay, MANN-WHITNEY

Journal: Molecular Oncology

Article Title: Long noncoding RNA LINC01578 drives colon cancer metastasis through a positive feedback loop with the NF‐κB/YY1 axis

doi: 10.1002/1878-0261.12819

Figure Lengend Snippet: Blocking of NF‐κB signaling abolished the oncogenic roles of LINC01578 in colon cancer. (A) Cell viability of LINC01578 overexpressed and control DLD‐1 cells treated with DMSO or 5 µ m JSH‐23 was determined by Glo cell viability assay. (B) Cell proliferation of LINC01578‐overexpressed and control DLD‐1 cells treated with DMSO or 5 µ m JSH‐23 was determined by EdU assays. Scale bars, 100 µm. (C) Cell migration of LINC01578‐overexpressed and control DLD‐1 cells treated with DMSO or 5 µ m JSH‐23 was determined by transwell migration assays. Scale bars, 100 µm. (D) Cell invasion of LINC01578‐overexpressed and control DLD‐1 cells treated with DMSO or 5 µ m JSH‐23 was determined by transwell invasion assays. Scale bars, 100 µm. Data are shown as mean ± SD based on three independent experiments. ** P < 0.01, ns, not significant, by Student's t ‐test.

Article Snippet: The nuclear extracts from indicated cells were used to measure NF‐κB transcription factor DNA binding activities using the NF‐κB p50 Transcription Factor Assay Kit (Cat. ab207217; Abcam) and NF‐κB p65 Transcription Factor Assay Kit (Cat. ab133112; Abcam) following the manufacturer's protocols.

Techniques: Blocking Assay, Viability Assay, Migration

Journal: Molecular Oncology

Article Title: Long noncoding RNA LINC01578 drives colon cancer metastasis through a positive feedback loop with the NF‐κB/YY1 axis

doi: 10.1002/1878-0261.12819

Figure Lengend Snippet: LINC01578 forms a positive feedback loop with NF‐κB/YY1. (A) YY1 mRNA and protein levels in DLD‐1 cells transfected with LINC01578 overexpression vector were determined by qRT‐PCR and western blot. (B) YY1 mRNA and protein levels in LoVo cells infected with shRNAs targeted to LINC01578 were determined by qRT‐PCR and western blot. (C) YY1 mRNA and protein levels in DLD‐1 cells transfected with LINC01578 overexpression vector and treated with 5 µ m JSH‐23 were determined by qRT‐PCR and western blot. (D) YY1 mRNA and protein levels in LoVo cells infected with shRNAs targeted to LINC01578 and treated with 5 µ m JSH‐23 were determined by qRT‐PCR and western blot. (E) A schematic model of positive feedback loop between LINC01578 and NF‐κB/YY1. (F) Luciferase reporter assays for LINC01578‐overexpressed and control DLD‐1 cells transfected with luciferase reporter plasmids containing LINC01578 promoter. (G) Luciferase reporter assays for LINC01578‐depleted and control LoVo cells transfected with luciferase reporter plasmids containing LINC01578 promoter. (H) ChIP assays using p50 and p65 antibodies were carried out in LINC01578‐overexpressed and control DLD‐1 cells. The enrichment of LINC01578 promoter was determined by qPCR. (I) ChIP assays using p50 and p65 antibodies were carried out in LINC01578‐depleted and control LoVo cells. The enrichment of LINC01578 promoter was determined by qPCR. (J) ChIP assays using YY1 antibody were carried out in LINC01578‐overexpressed and control DLD‐1 cells. The enrichment of LINC01578 promoter was determined by qPCR. (K) ChIP assays using YY1 antibody were carried out in LINC01578‐depleted and control LoVo cells. The enrichment of LINC01578 promoter was determined by qPCR. Data are shown as mean ± SD based on three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant, by Student's t ‐test (A,C,F,H,J), or one‐way ANOVA followed by Dunnett's multiple comparisons test (B,D,G,I,K).

Article Snippet: The nuclear extracts from indicated cells were used to measure NF‐κB transcription factor DNA binding activities using the NF‐κB p50 Transcription Factor Assay Kit (Cat. ab207217; Abcam) and NF‐κB p65 Transcription Factor Assay Kit (Cat. ab133112; Abcam) following the manufacturer's protocols.

Techniques: Transfection, Over Expression, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Infection, Luciferase