Journal: PLoS ONE

Article Title: Networked T Cell Death following Macrophage Infection by Mycobacterium tuberculosis

doi: 10.1371/journal.pone.0038488

Figure Lengend Snippet: (A) Addition of the pan-caspase inhibitor Z-VAD-FMK mitigated cell death induced by low MOI and high MOI-infected macrophage supernatants (shaded bars, infection + and ++ respectively), as well as background cell death observed in the uninfected control supernatants (closed bars, infection -). Cell death in infected supernatants was not mitigated by coincubation with blocking antibodies to TNF-α (B) or Fas (C), and in each case was significantly higher than in uninfected control supernatants (closed bars). Cell death induced by positive controls (D) (1 µM Staurosporine, 5 ng/ml recombinant human TNF-α +0.2 µg/ml cycloheximide and 10 ng/ml recombinant human Fas ligand) was in each case significantly reduced by coincubation with the corresponding inhibitor (open bars). Data shown in each panel are from one representative experiment, showing mean percentage cell death ± SEM from triplicate incubations. ** = p<0.01, *** = p<0.001, Student’s t test relative to uninfected control (A–C), or positive control relative to inhibitor (D).

Article Snippet: In positive control experiments for Fas ligand-mediated cell killing, cells were incubated in the presence of 10 ng/ml recombinant human Fas ligand (R&D systems), crosslinked with 10 μg/ml anti-6× Histidine (R&D systems).

Techniques: Infection, Control, Blocking Assay, Recombinant, Positive Control

Journal: Journal of Immunology Research

Article Title: Novel Application of Radotinib for the Treatment of Solid Tumors via Natural Killer Cell Activation

doi: 10.1155/2018/9580561

Figure Lengend Snippet: Radotinib enhances cytolytic activity of NK cells against Fas-expressing A549 cells. (a) Primary NK cells were isolated from healthy donors to perform NK cell cytotoxicity assay. The purity of CD3 − CD56 + NK cells was 96.6%. (b) To determine the effect of radotinib on the cytolytic activity of NK cells against A549 cells, the cells were treated with various concentrations of radotinib (0, 12.5, 25, 50, 100, and 200 μ M) for 48 h and the cytotoxicity assay was performed (E : T ratio = 5 : 1). (c) To determine if the effect of radotinib on NK cytotoxicity was mediated by the Fas receptor, Fas expression was transiently downregulated by Fas siRNA transfection into A549 cells. At approximately 70% confluency, A549 cells were incubated with 50 pmole Fas-specific siRNA or negative control siRNA using Lipofectamine RNAiMAX. Surface expression of Fas on A549 cells was determined by staining with PE-conjugated Fas antibody (solid line). PE-conjugated mouse IgG antibody was used as an isotype control (dotted line). (d) The effect of radotinib on NK cytolytic activity against Fas siRNA-transfected A549 cells was determined by cytotoxicity assay. Radotinib-treated NK cells were used as effector cells, and Fas siRNA-transfected A549 cells or control cells were used as target cells (E : T ratio = 2 : 1). All values were normalized relative to the control (radotinib 0 μ M). The relative level was set to 1 for the control. (e) To further confirm the involvement of Fas-Fas ligand interaction in the radotinib-enhanced NK cytotoxicity, recombinant human soluble Fas was used to block Fas ligand on NK cells. Various concentrations of soluble Fas were preincubated with resting NK cells or radotinib-treated NK cells for 1 h, and then cytotoxicity assays were performed (E : T ratio = 2 : 1). All values were normalized relative to the control (radotinib 0 μ M). The relative level was set to 1 for the control. Data are reported as mean ± SD. All values were analyzed by unpaired Student's t -tests using GraphPad Prism 5. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. All data presented are representative of three independent experiments.

Article Snippet: Commercially available human Fas siRNA and negative control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, CA, USA).

Techniques: Activity Assay, Expressing, Isolation, Cytotoxicity Assay, Transfection, Incubation, Negative Control, Staining, Control, Recombinant, Blocking Assay

Journal: PLOS One

Article Title: Differential expression and regulation of FASLG by miR-5195/miR-3941 in age-related hearing loss

doi: 10.1371/journal.pone.0331661

Figure Lengend Snippet: (A). Heat map illustrating differential expression data from RNA-Seq analysis. Heatmap of FASLG gene expression, represented as fold changes in mRNA levels, was generated for the ARHL (n = 12) and NH (n = 12); (B). Expression of FASLG was analyzed using RT-qPCR and confirmed through WB (NH: n = 12, ARHL: n = 14). (C). Expression of miR-5195 was detected using RT-qPCR in WB (NH: n = 6, ARHL: n = 5). (D). Expression of miR-3941 was detected using RT-qPCR in WB (NH: n = 14, ARHL: n = 12). U6 snRNA was used as an internal control (** p < 0.01). Abbreviations used are as follows: Normal hearing group (NH), Age-related hearing loss (ARHL), Whole blood (WB), Fas ligand ( FASLG ).

Article Snippet: For the transfection experiments, the FASLG Human 3’ UTR Clone (#SC210991, OriGene, Rockville, Maryland, USA), miR-5195 (HmiR1468 – MR04, Genecopoeia, Rockville, Maryland, USA), and miR-3941 (HmiR1052 – MR04, Genecopoeia, Rockville, Maryland, USA) were used.

Techniques: Quantitative Proteomics, RNA Sequencing, Gene Expression, Generated, Expressing, Quantitative RT-PCR, Control

Journal: PLOS One

Article Title: Differential expression and regulation of FASLG by miR-5195/miR-3941 in age-related hearing loss

doi: 10.1371/journal.pone.0331661

Figure Lengend Snippet: (A). Identification of 3′-UTR sequences in the FASLG gene predicted to include binding sites for miR-3941; (B). Identification of 3′-UTR sequences in the FASLG gene predicted to harbor binding sites for miR-5195-5p; (C). Dual luciferase reporter assay results for miR-3941, miR-5195, and their corresponding target 3’-UTR of FASLG in HEI-OC-1 cells; (D). Dual luciferase assay results of miR-3941, miR-5195 and their corresponding target 3’-UTR of FASLG in HeLa cell; The significant differences between FASLG and the miRNA target group are represented as ** p < 0.01. Abbreviations used are as follows: Fas ligand ( FASLG ), House Ear Institute-Organ of Corti 1 (HEI-OC-1).

Article Snippet: For the transfection experiments, the FASLG Human 3’ UTR Clone (#SC210991, OriGene, Rockville, Maryland, USA), miR-5195 (HmiR1468 – MR04, Genecopoeia, Rockville, Maryland, USA), and miR-3941 (HmiR1052 – MR04, Genecopoeia, Rockville, Maryland, USA) were used.

Techniques: Binding Assay, Luciferase, Reporter Assay

Journal: PLOS One

Article Title: Differential expression and regulation of FASLG by miR-5195/miR-3941 in age-related hearing loss

doi: 10.1371/journal.pone.0331661

Figure Lengend Snippet: (A). The light blue rectangular node represents the FASLG gene, while the light blue circular nodes represent the 35 selected miRNAs. The nodes with red text indicate the two miRNAs of interest, miR-3941 and miR-5195-5p. The arrows connecting FASLG to the miRNAs indicate that the thickness of the arrow corresponds to the context++ score of the miRNA, with thicker arrows representing lower scores. (B). We utilized blood samples from individuals with ARHL and found that A notable rise in FASLG expression was observed. Additionally, we identified miR-3941 and miR-5195 as potential regulators of FASLG expression. These miRNAs may bind to the 3’ UTR of the elevated FASLG , leading to down-regulation of gene expression. Abbreviations used are as follows: Fas ligand ( FASLG ), Age-related hearing loss (ARHL), Fas receptor (FAS-R), 3’untranslated region (3’UTR).

Article Snippet: For the transfection experiments, the FASLG Human 3’ UTR Clone (#SC210991, OriGene, Rockville, Maryland, USA), miR-5195 (HmiR1468 – MR04, Genecopoeia, Rockville, Maryland, USA), and miR-3941 (HmiR1052 – MR04, Genecopoeia, Rockville, Maryland, USA) were used.

Techniques: Expressing, Gene Expression

Journal: Journal of Oral Pathology & Medicine

Article Title: The PI3K Inhibitor HCD Promotes Caspase Activation in Head and Neck Squamous Cells by Upregulating the Extrinsic and Intrinsic Apoptosis Pathways

doi: 10.1111/jop.70060

Figure Lengend Snippet: HCD activates the death receptor‐mediated apoptotic signaling pathway in SCC cells. (A) SCC25 cells were incubated with HCD (IC 50 ) for 24, 48 and 72 h, after which the gene expression levels of death receptor‐associated apoptosis pathway were determined by RT‐PCR analysis. Treating SCC25 cells with (+) or without HCD (IC 50 ) for 72 h, and the protein expressions of TNF‐α, TNF‐R1, TNF‐R2, FasL, Fas, TRADD and FADD were assessed by western blotting (B), immunofluorescence microscopy (C) and flow cytometry analysis (D). β‐Actin was measured as the quality control. The blot was cut after transfer to allow separate probing with anti‐β‐Actin and anti‐target protein antibodies. The splice is indicated by a black line. Histograms represent statistical analysis of the relative expression levels. The nuclei were visualized by staining DNA with Hoechst 33342 (blue) and using a fluorescent microscope (200×). Arrowheads indicate cells feature. Nuclei were visualized by staining DNA with Hoechst 33342 (blue) and certain protein expressions were stained with FITC (green) using a fluorescent microscope (200×). Arrowheads indicate apoptotic cells expressing a certain protein. The percentage as defined by fluorescent stained protein expression levels of cells and compared to control cells by flow cytometry. All data are presented as mean ± SD from three independent biological replicates ( n = 3 per condition). Statistical significance was determined using appropriate tests, with * p < 0.01 and p < 0.05 indicating significant differences compared to DMSO (vehicle control)‐treated cells.

Article Snippet: The cells were then probed with specific antibodies against cyto. c (Santa Cruz Biotechnology, sc‐13560, RRID: AB_627383, 1:200), Bax (Santa Cruz Biotechnology, sc‐20067, RRID: AB_626726, 1:200), Bcl‐2 (Santa Cruz Biotechnology, sc‐7382, RRID: AB_626736, 1:200), TNF‐α (Santa Cruz Biotechnology, sc‐33639, RRID: AB_628371, 1:200), TNF‐R1 (Santa Cruz Biotechnology, sc‐1067, RRID: AB_632519, 1:200), TNF‐R2 (Santa Cruz Biotechnology, sc‐1074, RRID: AB_2206681, 1:200), FasL (Santa Cruz Biotechnology, sc‐834, RRID: AB_2100640, 1:200), Fas (Santa Cruz Biotechnology, sc‐8009, RRID: AB_627218, 1:200), TRADD (Santa Cruz Biotechnology, sc‐46653, RRID: AB_2209061, 1:200), FADD (Santa Cruz Biotechnology, sc‐5559, RRID: AB_2209061, 1:200), Caspase‐9 (Santa Cruz Biotechnology, sc‐17784, RRID: AB_673489), cleaved Caspase‐8 (Cell signaling technology, #9496, RRID: AB_561381, 1:1000), cleaved Caspase‐3 (Cell signaling technology, #9661, RRID: AB_2341188, 1:1000), and β‐actin (Santa Cruz Biotechnology, sc‐47 778, RRID: AB_2714189, 1:500) as outlined in a previously described protocol.

Techniques: Incubation, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunofluorescence, Microscopy, Flow Cytometry, Control, Expressing, Staining