Journal: The Journal of investigative dermatology

Article Title: Functional characterization of IL-17F as a selective neutrophil attractant in psoriasis.

doi: 10.1038/jid.2008.294

Figure Lengend Snippet: Figure 1. IL-17F induces IL-8 production by NHEKs. (a) IL-8 gene expression by IL-17F in NHEKs. Total RNA was extracted from the cell lysates 4 and 24 hours after stimulation with 100 ng ml1 IL-17F, 100 ng ml1 TNF-a, 100 ng ml1 IL-17A, and PBS control. The levels of mRNA for IL-8 were calculated as the fold induction compared with the PBS control using real- time PCR. (b) Analysis of IL-8 production in NHEKs stimulated with IL-17F (100 ng ml1), TNF-a (100 ng ml1), IL-17A (100 ng ml1), and PBS control. IL-8 protein release in the supernatant was determined by ELISA as described in ‘‘Materials and Methods’’. Results represent mean±SEM from at least three independent experiments. *Po0.05 was considered significant.

Article Snippet: In some experiments described below, rmTNF-a (100 ng in 50 ml of PBS), rm IL-17A (100 ng in 50 ml of PBS), rm IL-17F plus anti-human IL-8 antibody (100 ng rm IL-17F and 100 ng antihuman IL-8 Ab in total 50 ml of PBS) (all R&D Systems) were also intradermally administrated.

Techniques: Gene Expression, Control, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: The Journal of investigative dermatology

Article Title: Functional characterization of IL-17F as a selective neutrophil attractant in psoriasis.

doi: 10.1038/jid.2008.294

Figure Lengend Snippet: Figure 2. IL-8 gene expression in the mouse skin induced by IL-17F. Ear tissue from the IL-17F-treated and PBS control mice were collected 24 hours following intradermal injection and total RNA was isolated from each ear specimen. The primer sequence was as follows: upstream 50-ATGGCTGGG ATTCACCTCAA-30, downstream 50-AAGCCTCGCGACCATTCTT-30. The expression of IL-8 mRNA 24 hours was 2.9-fold higher than that in the control group. Results represent mean±SEM from three independent experiments.

Article Snippet: In some experiments described below, rmTNF-a (100 ng in 50 ml of PBS), rm IL-17A (100 ng in 50 ml of PBS), rm IL-17F plus anti-human IL-8 antibody (100 ng rm IL-17F and 100 ng antihuman IL-8 Ab in total 50 ml of PBS) (all R&D Systems) were also intradermally administrated.

Techniques: Gene Expression, Control, Injection, Isolation, Sequencing, Expressing

Journal: The Journal of investigative dermatology

Article Title: Functional characterization of IL-17F as a selective neutrophil attractant in psoriasis.

doi: 10.1038/jid.2008.294

Figure Lengend Snippet: Figure 3. Effect of inhibitors on IL-8 protein production in NHEKs. The cells were preincubated with varying concentrations of PD98059, U0126, SB202190, SP600125, or Me2SO vehicle for 1 hour, followed by stimulation with IL-17F (100 ng ml1) for 24 hours. Results represent mean±SEM from three independent experiments. *Po0.05 was considered significant versus IL-17F-stimulated cells without addition of inhibitors.

Article Snippet: In some experiments described below, rmTNF-a (100 ng in 50 ml of PBS), rm IL-17A (100 ng in 50 ml of PBS), rm IL-17F plus anti-human IL-8 antibody (100 ng rm IL-17F and 100 ng antihuman IL-8 Ab in total 50 ml of PBS) (all R&D Systems) were also intradermally administrated.

Techniques:

Journal: The Journal of investigative dermatology

Article Title: Functional characterization of IL-17F as a selective neutrophil attractant in psoriasis.

doi: 10.1038/jid.2008.294

Figure Lengend Snippet: Figure 4. Treatment with IL-17F induces ERK1/2 phosphorylation in the mouse skin. Ear tissue samples collected at 0.5, 1, and 2 hours following IL-17F injection (100 ng in 50 ml of PBS) were prepared. Western blot analysis of phosphorylated and total ERK1/2 was performed on these skin samples. The activation of ERK1/2 was seen at 0.5, 1, and 2 hours after injection. The results shown are representative of three separate experiments.

Article Snippet: In some experiments described below, rmTNF-a (100 ng in 50 ml of PBS), rm IL-17A (100 ng in 50 ml of PBS), rm IL-17F plus anti-human IL-8 antibody (100 ng rm IL-17F and 100 ng antihuman IL-8 Ab in total 50 ml of PBS) (all R&D Systems) were also intradermally administrated.

Techniques: Phospho-proteomics, Injection, Western Blot, Activation Assay

Journal: The Journal of investigative dermatology

Article Title: Functional characterization of IL-17F as a selective neutrophil attractant in psoriasis.

doi: 10.1038/jid.2008.294

Figure Lengend Snippet: Figure 5. Histologic examination. Histopathological findings 48 hours after intradermal injection of (a) IL-17F, (c) TNF-a, (d) IL-17A, and (e) PBS control. Bar ¼ 100 mm. (b) High magnification of a. Infiltrating cells in the dermis consisted mainly of neutrophils (arrow). Bar ¼ 50 mm. (g) A cell count was performed as described in ‘‘Materials and Methods’’, and the density of dermal infiltration was expressed as mononuclear cells, neutrophils, eosinophils, and total number of cells. No infiltrating eosinophils were seen in both the IL-17F-treated and other groups. The number of neutrophils in the dermis was significantly increased by injection of recombinant mouse IL-17F compared with the control group (*Po0.05) and the inflammatory cells in the dermis were significantly but not completely blocked by anti-IL-8 antibody (*Po0.05) (f, g). Bar ¼ 100 mm.

Article Snippet: In some experiments described below, rmTNF-a (100 ng in 50 ml of PBS), rm IL-17A (100 ng in 50 ml of PBS), rm IL-17F plus anti-human IL-8 antibody (100 ng rm IL-17F and 100 ng antihuman IL-8 Ab in total 50 ml of PBS) (all R&D Systems) were also intradermally administrated.

Techniques: Injection, Control, Cell Counting, Recombinant

Journal: The Journal of investigative dermatology

Article Title: Functional characterization of IL-17F as a selective neutrophil attractant in psoriasis.

doi: 10.1038/jid.2008.294

Figure Lengend Snippet: Figure 6. IL-17F protein in the skin of psoriasis patients. (a) Representative results from analysis of IL-17F protein production in the nonlesional and lesional skin of psoriasis patients. Skin specimens were homogenized and the supernatants were examined by western blotting. (b) Protein level of IL-17F in the psoriatic lesional skin was higher than in nonlesional psoriatic skin (*Po0.05). Low level of IL-17F protein was also detected in nonlesional psoriatic skin. The results were expressed as the mean±SEM (n ¼ 5). *Po0.05 was considered significant.

Article Snippet: In some experiments described below, rmTNF-a (100 ng in 50 ml of PBS), rm IL-17A (100 ng in 50 ml of PBS), rm IL-17F plus anti-human IL-8 antibody (100 ng rm IL-17F and 100 ng antihuman IL-8 Ab in total 50 ml of PBS) (all R&D Systems) were also intradermally administrated.

Techniques: Western Blot

Journal: Nature immunology

Article Title: Syndromic immune disorder caused by a viable hypomorphic allele of spliceosome component Snrnp40

doi: 10.1038/s41590-019-0464-4

Figure Lengend Snippet: a-j, Mice were infected with MCMV (a,c,e-j) or HSV-1 (b,d). a, MCMV titers in the spleens (left) and livers (right) five days after MCMV infection (n=9 mice for +/+ and swp/+, n=8 mice for and rplc/+ and −/+, n=7 mice for swp/swp, n=3 mice for swp/rplc). Statistical significance was determined by one-way ANOVA with Sidak’s multiple comparisons using Log10 of viral titers. ****P<0.0001. b, HSV-1 titers in the spleens (left) and livers (right) seven days after HSV-1 infection (n=8 mice per genotype). Statistical significance was determined by one-way ANOVA with Sidak’s multiple comparisons using Log10 of viral titers. **P=0.0084, **P=0.0063, *P=0.048. c, Survival curves after MCMV infection (n=8 mice per genotype; ****P<0.0001, two-sided Log-rank (Mantel-Cox) test). The experiment was concluded after 7 days, but no additional death was observed for at least 10 additional days. d, Survival curves after HSV-1 infection (n=8 mice per genotype; **P=0.0011, two-sided Log-rank test). e-j, Serum concentrations of IL-6 (e), IL-12 p70 (f), TNF (g), IFN-β (h), IFN-γ (i), and IL-17A (j) 36 h after MCMV infection (n=8 mice per genotype; the indicated P values were determined by unpaired, two-tailed Student’s t-test). k,l, Equal numbers of CellTrace™ Violet-labeled C57BL/6J splenocytes (control cells) and B2m-KO splenocytes (NK target cells) were injected i.v. into recipient mice of the indicated genotypes. k, Flow cytometry of control and NK target cells remaining in the blood of recipient mice 3 days after injection. Numbers indicate percent of injected control and NK target cells. l, Quantification of the percentage of NK target cells killed in the recipient mice (n=18 mice for +/+, n=15 mice for swp/swp; ****P<0.0001, two-way ANOVA with Sidak’s multiple comparisons. Data are representative of two (b-d), three (a,e-j), or four (k,l) independent experiments (line indicates median in a,b; mean ± s.d. in e-j,l).

Article Snippet: Antibodies Primary antibodies used for immunoblotting were: anti-Snrnp40 (HPA026527) from Atlas; anti-FLAG M2 (F1804) from Sigma-Aldrich; anti-Themis (06–1328) and anti-GAPDH (MAB374) from Millipore; anti-Snrnp200 (A303–453A-T) from Bethyl; anti-Eftud2 (10208–1-AP) from Proteintech; anti-Cd2bp2 (PA5–18286) from Invitrogen; anti-Ikzf3 (Aiolos, 15103), anti-IL-17A (13838), anti-IL-17F (13186), anti-α-tubulin (3873), anti-β-actin (3700), anti-Hsp90 (4874) and anti-GFP (2956) from Cell Signaling.

Techniques: Infection, Two Tailed Test, Labeling, Injection, Flow Cytometry

Journal: Nature immunology

Article Title: Syndromic immune disorder caused by a viable hypomorphic allele of spliceosome component Snrnp40

doi: 10.1038/s41590-019-0464-4

Figure Lengend Snippet: a, Heatmap for differentially expressed immune genes in unstimulated or PMA/ionomycin stimulated Snrnp40-KO and wild-type EL4 cells. Data represent Log2 of fold change vs. unstimulated WT. b, Venn diagram for 655 unique differentially expressed genes. 30.0% (197) were affected in both unstimulated and stimulated conditions. c, RT-qPCR analysis of the indicated mRNAs in Snrnp40-KO and wild-type EL4 cells (n=3 independent cell lines per group; ***P=0.00063, **P=0.0024, **P=0.0023, **P=0.0043, *P=0.014, **P=0.0014, *P=0.01, **P=0.0062, *P=0.044, unpaired, two-tailed Student’s t-test). d, RT-qPCR analysis of the indicated mRNAs in Snrnp40swp/+ and Snrnp40swp/rplc splenocytes (n=3 mice per genotype; **P=0.0080, **P=0.0025, **P=0.0058, *P=0.021, *P=0.022, *P=0.039, **P=0.0078, unpaired, two-tailed Student’s t-test). e, RT-qPCR analysis of Ikzf3 in Snrnp40swp/+ and Snrnp40swp/rplc bone marrow (n=6 mice per genotype; ****P<0.0001, unpaired, two-tailed Student’s t-test). f,g, Immunoblot analysis of Ikzf3 expression in bone marrow (f) and Themis expression in thymocytes (g) of Snrnp40swp/+ and Snrnp40swp/rplc mice. h, RT-qPCR analysis of Il17a and Il17f in unstimulated (US) and PMA/ionomycin (50 ng/ml PMA and 1 μg/ml ionomycin) stimulated Snrnp40-KO and wild-type EL4 cells (n=3 independent cell lines per group; ***P=0.00045, **P=0.0073, *P=0.041, *P=0.016, unpaired, two-tailed Student’s t-test). i, Immunoblot analysis of IL-17A and IL-17F expression in Snrnp40-KO and wild-type EL4 cells at indicated time points after PMA/ionomycin stimulation. j,k, Concentration of IL-17A (j) and IL-17F (k) in the supernatants of Snrnp40-KO and wild-type EL4 cells (n=3 independent cell lines per group) at different time points after PMA/ionomycin stimulation. ****P<0.0001, two-way ANOVA with Sidak’s multiple comparisons test). RNA-seq experiment was performed one time (a,b). Data are representative of two (c-e,j,k) or three (f-i) independent experiments (mean ± s.d. in c-e,h,j,k). Quantities of mRNA are expressed relative to Gapdh transcript level (c-e,h). The blots (f,g,i) were cropped to show relevant bands and their original images are presented in the Source Data.

Article Snippet: Antibodies Primary antibodies used for immunoblotting were: anti-Snrnp40 (HPA026527) from Atlas; anti-FLAG M2 (F1804) from Sigma-Aldrich; anti-Themis (06–1328) and anti-GAPDH (MAB374) from Millipore; anti-Snrnp200 (A303–453A-T) from Bethyl; anti-Eftud2 (10208–1-AP) from Proteintech; anti-Cd2bp2 (PA5–18286) from Invitrogen; anti-Ikzf3 (Aiolos, 15103), anti-IL-17A (13838), anti-IL-17F (13186), anti-α-tubulin (3873), anti-β-actin (3700), anti-Hsp90 (4874) and anti-GFP (2956) from Cell Signaling.

Techniques: Quantitative RT-PCR, Two Tailed Test, Western Blot, Expressing, Concentration Assay, RNA Sequencing Assay

Journal: PLoS ONE

Article Title: T Cells Induce Pre-Metastatic Osteolytic Disease and Help Bone Metastases Establishment in a Mouse Model of Metastatic Breast Cancer

doi: 10.1371/journal.pone.0068171

Figure Lengend Snippet: CD3 + T cells derived from iliac BM of BALB/c mice, 11 days after 4T1 (T 4T1) or 67NR(T 67NR) tumor cells injection into the mammary fat pad, or control T cells from naïve mice (T Nv) were transferred intravenously to athymic nude mice and challenged with the soluble fraction of tumor antigen lysate (sAg). ( A ) 14 days after transference, spleen cells were restimulated with sAg and IL-17F and RANKL production was evaluated by ELISA. Data are expressed as the mean ± SD of five mice/group and are representative of two independent experiments * p≤0.05. ( B ) Frequency of IL-17F + RANKL + T cells was assessed by flow cytometry, 14 days after T cells transference. Plots show data from CD3 + CD4 + gated T cells. ( C ) Serum concentrations of OPG and RANKL and the OPG/RANKL ratio, measured by ELISA, 14 days after T cells transference. Data are expressed as the mean ± SD of five mice/group. a, b, c ,d** p<0.001 . ( D ) Histomorphometric analysis of the iliac bones from mice of the different groups and ( E ) high resolution µCT analysis of the iliac bones. Both analyses were performed as described in . Results shown are representative of at least two independent experiments with 5 mice/group. a, b, c ,d* p<0.05.

Article Snippet: Freshly isolated femur BM cells from BALB/c mice (6–8 weeks old) were cultured at a density of 1x10 5 cells per well, in 24-well plates, in DMEM plus 10% FBS, containing supernatants from sAg stimulated iliac BM cells in the presence of M-CSF (10ng/mL), with or without recombinant OPG (10ng/mL) (Peprotech) or rat anti-mouse IL-17F mAb (10ng/mL) (R&D systems), for 7 days, at 37 ° C. Positive controls received recombinant RANKL (10ng/mL) (Peprotech).

Techniques: Derivative Assay, Injection, Control, Enzyme-linked Immunosorbent Assay, Flow Cytometry