all cytokines were supplied  (PeproTech)

Journal: Science Advances

Article Title: cIAP1/2 inhibition synergizes with TNF inhibition in autoimmunity by down-regulating IL-17A and inducing T regs

doi: 10.1126/sciadv.aaw5422

Figure Lengend Snippet: ( A ) Chemical structure of the SMAC mimetic, compound GT13072. ( B ) Mice with CIA received daily intraperitoneal injections of GT13072 or control compound at 10 mg/kg per day. ( C ) All four paws were assessed daily for signs of arthritis. P values were calculated using two-way analysis of variance (ANOVA) with Tukey’s post hoc test and are not shown in the graph for clarity. ( D ) Histological analysis of arthritic paws from mice with CIA after 10 days of treatment with GT13072 or control compound ( n = 10). ( E ) On day 10 of arthritis, serum samples from CIA mice were analyzed for cytokines by Meso Scale Discovery (MSD). Each data point represents one animal ( n = 19 to 22). Error bars represent ±SEM. The horizontal lines represent the mean. P values were calculated using unpaired Student’s t tests. ( F to I ) GT13072-treated and control CIA mice were culled on day 10, and their draining lymph node and arthritic paw cells were counted and characterized by flow cytometry. P values were calculated using unpaired Student’s t tests. Each data point represents one animal (lymph node, n = 19 to 22; paw, n = 10). (F and G) The percentage of IL-17A–secreting CD4 + T cells in lymph nodes and paws was determined by flow cytometry. One representative dot blot or contour plot is shown. Absolute numbers of CD4 + IL-17A + cells in lymph nodes and arthritic paws are shown. (H) Absolute numbers of TCRγδ cells and IL-17 + TCRγδ T cells in arthritic paws with representative contour plot. (I) Paw cells were examined for RORγT expression.

Article Snippet: All cytokines were supplied by PeproTech, except IL-23 (R&D Systems).

Techniques: Flow Cytometry, Dot Blot, Expressing

Journal: Science Advances

Article Title: cIAP1/2 inhibition synergizes with TNF inhibition in autoimmunity by down-regulating IL-17A and inducing T regs

doi: 10.1126/sciadv.aaw5422

Figure Lengend Snippet: ( A ) cIAP1 and cIAP2 protein expression in human CD4 + lymphocytes cultured for 10 min with GT13072 (1000 nM) or control compound. Immunoblots are representative of three independent experiments. ( B and C ) Human CD4 + CCR6 + CD161 + T cells were cultured in the presence of T H 17-differentiating cytokines and anti-CD3/CD28 Dynabeads for 6 days. The effect of GT13072 or control compound (1000 nM) on cytokine secretion and RORγT was determined by flow cytometry ( n = 4). Representative dot blots from four donors are shown. Bar graphs represent quantification of flow cytometry data. ( D ) Naïve human CD4 + T cells were cultured in the presence of T H 17-differentiating cytokines and anti-CD3/CD28 Dynabeads plus GT13072 or control compound (1000 nM). After 17 hours, mRNA levels were determined by quantitative reverse transcription polymerase chain reaction. Data are expressed as the mRNA level normalized to RPLPO expression and are shown as means ± SEM from five independent human donors. RPLPO, ribosomal protein, large, P0. (B to D) Error bars represent ±SEM. P values were calculated using unpaired Student’s t tests.

Article Snippet: All cytokines were supplied by PeproTech, except IL-23 (R&D Systems).

Techniques: Expressing, Cell Culture, Western Blot, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction

Journal: Science Advances

Article Title: cIAP1/2 inhibition synergizes with TNF inhibition in autoimmunity by down-regulating IL-17A and inducing T regs

doi: 10.1126/sciadv.aaw5422

Figure Lengend Snippet: ( A ) Phosphorylation of p38 MAPK was determined in human CD4 + T cells cultured with 1000 nM GT13072 or control compound before stimulation with TNF-α at 20 ng/ml. Immunoblots are representative of two independent experiments. p, protein. ( B and C ) Naïve human CD4 + T cells cultured with 1000 nM GT13072 before stimulation with plate-bound anti-CD3 (5 μg/ml), soluble anti-CD28 (2 μg/ml), IL-6 (50 ng/ml), IL-1β (10 ng/ml), and transforming growth factor–β (TGF-β; 1 ng/ml) for the indicated durations. (B) Phospho-p38 MAPK detected by immunoblotting. Results are representative of two independent experiments. (C) NFATc1 protein detected by immunoblotting. Dotted line shows where lanes were removed from the blot. Results are representative of four independent experiments. ( D and E ) Human CD4 + CCR6 + CD161 + T cells were cultured in the presence of T H 17-differentiating cytokines and anti-CD3/CD28 Dynabeads for 6 days. (D) Total number of cells on day 6 of culture. (E) Determination of annexin V and propidium iodide (PI) expression on day 6. Representative dot blots are shown from three donors.

Article Snippet: All cytokines were supplied by PeproTech, except IL-23 (R&D Systems).

Techniques: Cell Culture, Western Blot, Expressing

Journal: Science Advances

Article Title: cIAP1/2 inhibition synergizes with TNF inhibition in autoimmunity by down-regulating IL-17A and inducing T regs

doi: 10.1126/sciadv.aaw5422

Figure Lengend Snippet: ( A to C ) Mice with CIA received injections of GT13072 alone, etanercept alone, and GT13072 plus etanercept until day 10. All treatment was stopped on day 10 of arthritis. Animals were culled on day 18 of arthritis ( n = 7). (B) Clinical scores from day 10 to day 18. Error bars represent ±SEM. Representative photomicrographs of arthritic paws from mice on day 18. (C) Human RA patient synovial cells were cultured in the presence of T H 17-differentiating cytokines and anti-CD3/CD28 Dynabeads for 6 days. The effect of GT13072 or control compound (1000 nM) on IL-17A and RORγT was determined by flow cytometry.

Article Snippet: All cytokines were supplied by PeproTech, except IL-23 (R&D Systems).

Techniques: Cell Culture, Flow Cytometry