Journal: Molecular Systems Biology

Article Title: Modeling signaling‐dependent pluripotency with Boolean logic to predict cell fate transitions

doi: 10.15252/msb.20177952

Figure Lengend Snippet: Summation of frequencies (0–1) of O/S/N‐positive cells assessed by high content screening at 2 days after medium replacement ( n = 6 for Oct4 and Sox2, n = 4 for Nanog; the error bars represent s.d.). Asterisk indicates the significant difference for total OSN assessed by unpaired, two‐sided student t ‐test (** P < 0.05, * P < 0.1). Upper panel: A measure of Pearson's correlation (PCC) for OSN in 2i‐supplemented conditions, quantified using high content screening ( n = 6 for Oct4 and Sox2, n = 4 for Nanog; the error bars represent s.d.). Lower panel: A measure of PCC for OSN in the results of a minimal perturbation‐sensitivity analysis described in (C). The significance was assessed by Wilcoxon exact rank test (* P < 0.05, ** P < 0.01). The results of minimal perturbation‐sensitivity analysis of the model where the model network was perturbed by removing single regulatory edges. Purple indicates down‐regulation of the genes by removing the regulatory edge, which means the regulation had a positive role on the expression level of O/S/N, while green indicates the reverse. Note that the results shown are the effects of regulatory edge removal: the removal of inhibitory regulation from Cdx2 to Oct4 increases Oct4, which means that the regulation edge acts as a negative effector for Oct4 level. The difference in PSC population stability between 2iL and 2i−L conditions was assessed via published single gene LOF and double genes LOF in vitro (Dunn et al , ; EXP) and in silico (SIM). The upper panel depicts the experimental results for the relative count of alkaline phosphatase (AP) positive cells to untreated colonies upon gene manipulations. The simulation data (lower panel) shows population‐averaged expression level of Oct4 relative to controls (2iL and 2i−L) where the manipulated gene was set as continuously OFF. Blue nodes indicate the response in 2i−L, while red nodes indicate those in 2iL ( n = 5 for simulation and n = 3–5 for experimental). The experimental inputs (additives of the conditions) corresponding to the simulation inputs shown in Fig E. Abbreviations used are as follows: Jaki for Janus kinase (JAK) inhibitor, Dkk1 for Dickkopf1, and Alki for chemical inhibitor selective for Activin receptor‐like kinase (ALK) 4/5/7. Predicted levels (left) and measured gene‐expressing cell frequencies (right) of O/S/N for each condition group (L, LIF; W, WNT). The average value of four distinct signal conditions (±BMP±Activin/Nodal) in each group for each gene was calculated and summed up into an OSN score ( n = 6 for Oct4 and Sox2, n = 4 for Nanog; the error bars represent s.d.). Asterisk indicates the significant difference for total OSN assessed by Wilcoxon exact rank test (** P < 0.05, * P < 0.1). Susceptibility of O/S/N expression levels of PSCs to Activin and BMP signal perturbations was predicted (upper panel) and measured (lower panel). Standard deviation per mean of predicted expression levels or gene‐expressing frequencies from immunostaining was calculated as a coefficient of variation for each group (±LIF±WNT) including four distinct signal conditions (±BMP±Activin/Nodal). Frequencies of O/S/N‐expressing cells in the 19 combinatorial signal conditions with and without serum assessed by immunostaining ( n = 2). Most conditions were equivalent except for −L–W−B+A and +L−W−B−A. The notable down‐regulation of OSN in −L+W+B−A (=2iJ+B−A) was seen in both with‐ and without‐serum conditions. The error bars represent s.d. of five independent simulations. Source data are available online for this figure.

Article Snippet: Validation of predicted responses to exogenous signaling was performed in serum‐containing medium supplemented with combinations of the following cytokines/small molecules: LIF (Millipore ESG1107—10 ng/ml), JAK inhibitor (EMD Millipore 420097—2.0 μsM), BMP4 (R&D Systems 314‐BP‐010—10 ng/ml), LDN193189 (Reagents Direct 36‐F52—0.1 μM), CHIR99021 (Reagents Direct 27‐H76—3 μM), Dkk1 (R&D Systems 1765‐DK‐010—275 ng/ml), bFGF (Peprotech 100‐18B—20 ng/ml), PD0325901 (Reagents Direct 39‐C68—1 μM), Activin A (R&D Systems 338‐AC‐050—20 ng/ml), and ALK5 inhibitor II (Enzo Life Sciences ALX‐270‐445—10 μM and Cedarlane ALX‐270‐445 for RNA‐seq).

Techniques: High Content Screening, Expressing, In Vitro, In Silico, Standard Deviation, Immunostaining

Journal: Frontiers in Immunology

Article Title: Natural Killer T Cell-Targeted Immunotherapy Mediating Long-term Memory Responses and Strong Antitumor Activity

doi: 10.3389/fimmu.2017.01206

Figure Lengend Snippet: The in vivo adjuvant effects of RK-pulsed dendritic cells (DCs). (A) In vivo expansion of natural killer T (NKT) cells upon injection of RK-DCs. Flow cytometry of wild-type B6 splenocytes 6 days after intravenous injection of 5 × 10 4 unpulsed DCs or RK-DCs. Numbers indicate percentages of CD1d dimer + TCRβ + NKT cells within the 7-AAD − CD8 − CD19 − viable splenocyte gate. Data are representative of 3 mice per group. (B) Absolute numbers of splenic NKT cells as shown in panel (A) . Data are mean ± SEM from three mice per group. (C) Intracellular analysis of IFN-γ production by splenic NK cells. Unpulsed- or RK-pulsed DCs (5 × 10 4 per mouse) were injected intravenously into B6 mice. Splenocytes were isolated after 16 h and cultured in complete media containing GolgiPlug (BD Biosciences) for 1 h. Numbers on flow cytometry plots show percentages of NK1.1 + IFN-γ + cells among NK1.1 + CD3ε − gated cells. The data are representative of n = 3 mice per group. (D) Absolute cell numbers of IFN-γ + NK cells. Data are mean ± SEM from n = 3 mice per group. (E) Detection of OVA-specific tetramer-positive CD8 effector T cells. B6 mice were immunized with OVA antigen together with unpulsed- or RK-pulsed DCs (5 × 10 4 per mouse) on day 0 and liver mononuclear cells were analyzed 7 days later. Numbers on flow cytometry plots indicate percentages of OVA tetramer-positive cells among viable CD8 T cells. (F) Absolute numbers of OVA tetramer + CD8 T cells (mean ± SEM, n = 3 mice per group) gated as shown in panel (E) . Experiments shown in panels (A–F) were repeated two times with similar results. (G) Detection of IL-12p70 in the sera collected 6 h after intravenous injection of 5 × 10 4 unpulsed- or RK-pulsed DCs into B6 mice. Serum IL-12p70 levels were measured by cytometric bead array. Data are mean ± SEM from three mice, and experiments were repeated three times with similar results. (H) Detection of IL12B mRNA encoding IL-12p40 by real-time quantitative PCR. RK-pulsed human PBMNC-derived DCs (1 × 10 5 per well) were cultured for 12 h with or without recombinant human CD40 ligand. Bars depict the relative gene expression (mean ± SEM from triplicate wells) with GAPDH used as internal control. The experiment was repeated with three different donors with essentially similar results. *** P < 0.001, unpaired Student’s t -test; n.d., not detected.

Article Snippet: Human PBMNC-derived DCs (1 × 10 5 per well) were cultured in the presence or absence of histidine-tagged recombinant human CD40 Ligand (0.1 µg/mL; from R&D) and His Tag Antibody (10 µg/mL; from R&D) in 96-well culture plates for 12 h. RNA was purified using an RNeasy Plus Micro kit (Qiagen), and cDNA was prepared with Superscript VILO cDNA Synthesis Kit (Life Technologies).

Techniques: In Vivo, Injection, Flow Cytometry, Isolation, Cell Culture, Real-time Polymerase Chain Reaction, Derivative Assay, Recombinant, Expressing

Journal: Annals of the Rheumatic Diseases

Article Title: MICL controls inflammation in rheumatoid arthritis

doi: 10.1136/annrheumdis-2014-206644

Figure Lengend Snippet: Myeloid inhibitory C-type lectin-like receptor (MICL) is expressed on myeloid cells during human rheumatoid arthritis (RA). (A) Immunohistochemical analysis of expression of hMICL (brown) in haematoxylin-stained synovial tissue sections from three patients with RA (top panels) and non-inflamed synovium from a patient with osteoarthritis (bottom panels). (B) Immunohistochemical analysis of serial sections of RA synovium probed for CD163 or αhMICL, as indicated. Sections were counterstained with haematoxylin. Scale bar, 50 μm.

Article Snippet: For dot blots, serum samples were used to probe recombinant histidine-tagged hMICL or histidine-tagged human C-type lectin superfamily member 8 (ClecSF8; Creative Biomart), immobilised on nitrocellulose membranes and then detected with peroxidase-conjugated goat anti-human IgG F(ab′) 2 fragment (Jackson ImmunoResearch) or mouse anti-His monoclonal antibody (Qiagen).

Techniques: Immunohistochemical staining, Expressing, Staining

Journal: Annals of the Rheumatic Diseases

Article Title: MICL controls inflammation in rheumatoid arthritis

doi: 10.1136/annrheumdis-2014-206644

Figure Lengend Snippet: Myeloid inhibitory C-type lectin-like receptor (MICL) is an autoantigen in human rheumatoid arthritis (RA). (A) ELISA-based analyses of anti-hMICL reactivity in serum samples from patients with RA and healthy donors. (B) Dot-blot analysis of reactivity to his-tagged MICL (hMICL) and control proteins in two patients with RA and a normal control. (C) Exacerbation of collagen antibody-induced arthritis in wild-type mice after the intraperitoneal administration of anti-mMICL and isotype control monoclonal antibodies, as described in ‘Methods’. Shown are pooled data from two independent experiments (n=10). *p<0.05.

Article Snippet: For dot blots, serum samples were used to probe recombinant histidine-tagged hMICL or histidine-tagged human C-type lectin superfamily member 8 (ClecSF8; Creative Biomart), immobilised on nitrocellulose membranes and then detected with peroxidase-conjugated goat anti-human IgG F(ab′) 2 fragment (Jackson ImmunoResearch) or mouse anti-His monoclonal antibody (Qiagen).

Techniques: Enzyme-linked Immunosorbent Assay, Dot Blot, Control, Bioprocessing