Journal: iScience

Article Title: Pharmacological inhibition of RAS overcomes FLT3 inhibitor resistance in FLT3-ITD+ AML through AP-1 and RUNX1

doi: 10.1016/j.isci.2024.109576

Figure Lengend Snippet:

Article Snippet: CD217 (IL17RA) Antibody, anti-human, APC, REAfinity , Miltenyi Biotec , Cat# 130-127-293; RRID: AB_2904838.

Techniques: Control, Virus, Recombinant, Modification, Saline, Stripping Membranes, Protease Inhibitor, Reverse Transcription, Membrane, Labeling, Gel Extraction, Plasmid Preparation, RNA Library Preparation, Library Quantification, Software

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IL-17RA and IL-17RC receptors are essential for IL-17A-induced ELR+ CXC chemokine expression in synoviocytes and are overexpressed in rheumatoid blood.

doi: 10.4049/jimmunol.180.1.655

Figure Lengend Snippet: FIGURE 1. IL-17RA and IL-17RC mRNA expression in blood. A, IL-17RA and IL-17RC mRNA expression in peripheral whole blood from 40 RA patients (31 patients with severe RA and 9 with moderate RA) and from 19 healthy volunteers (HV), as deter- mined by microarray analysis. Results are presented as fluorescent intensity (, p 0.05; , p 0.005; , p 0.0005 by Mann-Whitney U test). B, IL-17RA or IL- 17RC were detected in whole blood from HV (n 3) or RA patients (n 6) by Western blotting using actin as a loading control. Den- sity ratios of IL-17RA or IL-17RC were nor- malized to actin and expressed as AU (, p 0.05 by Mann-Whitney test). C, IL-17RA and IL-17RC mRNA expression in peripheral blood subsets from three RA patients. CD14 monocytes, CD3 lymphocytes, and CD19 lymphocytes were isolated from PBMC by positive selection. IL-17RA and IL-17RC mRNA expression were assessed by real-time RT-PCR. PPIB was used to nor- malize gene expression. Values represent the mean SEM expression from three RA pa- tients (, p 0.05 by Mann-Whitney test).

Article Snippet: Human rIL-17A, the mAb against human IL-17RA and the polyclonal goat Ab against IL-17RC, were purchased from R&D Systems.

Techniques: Expressing, Microarray, MANN-WHITNEY, Western Blot, Control, Isolation, Selection, Quantitative RT-PCR, Gene Expression

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IL-17RA and IL-17RC receptors are essential for IL-17A-induced ELR+ CXC chemokine expression in synoviocytes and are overexpressed in rheumatoid blood.

doi: 10.4049/jimmunol.180.1.655

Figure Lengend Snippet: FIGURE 2. Detection of IL-17RA and IL-17RC in RA synovium. Im- munostaining of paraffin-embedded serial sections using mouse anti-IL- 17RA mAb (A and B, respectively, 200 and 400) and goat anti-IL- 17RC polyclonal Ab (C and D, respectively, 200 and 400) shows a diffuse positive staining (brown) in both the lining and sublining area. Control staining was performed using mouse IgG1 and goat serum (200) (insets in A and C). Immunostaining of IL-17RA and IL-17RC were also performed in OA synovium (E and F, respectively; 200). Some of the IL-17A-producing cells have a plasmacytoid-like appearance as suggested by the remote nucleus in a large cytoplasm (inset in D, 600).

Article Snippet: Human rIL-17A, the mAb against human IL-17RA and the polyclonal goat Ab against IL-17RC, were purchased from R&D Systems.

Techniques: Staining, Control, Immunostaining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IL-17RA and IL-17RC receptors are essential for IL-17A-induced ELR+ CXC chemokine expression in synoviocytes and are overexpressed in rheumatoid blood.

doi: 10.4049/jimmunol.180.1.655

Figure Lengend Snippet: FIGURE 4. Effect of IL-17RA siRNA and IL-17RC siRNA on IL-17A- or IL-17F-induced IL-6 and IL-8 secretion by RA synoviocytes. RA synovio- cytes were transfected with IL-17RA siRNA and IL-17RC siRNA at 0.5 and 0.05 g, respectively. A scrambled nonsilence siRNA, siCONTROL, was used as a negative control. A, Knockdown efficiency of IL-17RA siRNA and IL-17RC siRNA was assessed by real-time RT-PCR. The values represent data obtained 24 h posttransfection and are expressed as mean SEM of three independent experiments (, p 0.05 by Dunnett’s test). B, Knockdown efficiency of IL-17RA siRNA and IL-17RC siRNA was assessed by Western blotting 48 h posttransfection, using actin as a loading control. A representative western blot among three separate experiments is shown. C, Effect of IL-17RA siRNA and IL-17RC siRNA on IL-17A-induced IL-6 and IL-8 secretion by RA synoviocytes. Forty-eight hours after siRNA delivery (siCONTROL, IL-17RA siRNA, or IL-17RC siRNA), RA synoviocytes were stimulated for 12 h with IL-17A at 50 ng/ml or TNF- at 0.5 ng/ml. IL-6 and IL-8 levels were quantified in removed supernatants by ELISA. The values are expressed as fold induction compared with the untreated condition (mean SEM in untreated cells transfected with siCONTROL siRNA: 2.4 0.8 ng/ml and 367 39.6 pg/ml, respectively) and are represented the mean SEM of three independent experiments (, p 0.05 by Dunnett’s test).

Article Snippet: Human rIL-17A, the mAb against human IL-17RA and the polyclonal goat Ab against IL-17RC, were purchased from R&D Systems.

Techniques: Transfection, Negative Control, Knockdown, Quantitative RT-PCR, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IL-17RA and IL-17RC receptors are essential for IL-17A-induced ELR+ CXC chemokine expression in synoviocytes and are overexpressed in rheumatoid blood.

doi: 10.4049/jimmunol.180.1.655

Figure Lengend Snippet: FIGURE 5. Effect of IL-17RA and/or IL- 17RC inhibition by siRNA or extracellular inhib- itors, on IL-17 plus TNF--induced IL-6 secre- tion. A, Efficiency of posttranscriptional inhibition of IL-17RA and/or IL-17RC using siRNA. RA synoviocytes from the three cell pop- ulations (transfected with siCONTROL, IL- 17RA siRNA, and IL-17RC siRNA) were stim- ulation for 36 h with IL-17A (50 ng/ml), alone or in combination with TNF- (0.5 ng/ml). IL-6 levels were quantified in removed supernatants by ELISA. The values represent the mean SEM of three independent experiments (, p 0.05 by Dunnett’s test). B and C, Efficiency of extracellular inhibition using Abs. RA synovio- cytes were preincubated for 2 h (37°C, 5% CO2) with anti-IL-17RA Ab (10 g/ml) and/or anti- IL-17RC Ab (10 g/ml). The cells were then stimulated with IL-17A (50 ng/ml), alone or in combination with TNF- (0.5 ng/ml). IL-6 and CCL20 levels were quantified in removed super- natants by ELISA. The values represent the mean SEM of three independent experiments (mean SEM in untreated cells: 1.7 0.6 ng/ml and 1.4 1.6 pg/ml, respectively) (, p 0.05 by Dunnett’s test).

Article Snippet: Human rIL-17A, the mAb against human IL-17RA and the polyclonal goat Ab against IL-17RC, were purchased from R&D Systems.

Techniques: Inhibition, Transfection, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Th17-Derived Cytokines Synergistically Enhance IL-17C Production by the Colonic Epithelium.

doi: 10.4049/jimmunol.2200125

Figure Lengend Snippet: FIGURE 2. TNF-a stimulation does not prime cells to respond to IL-17A or IL-22. (A) Cells were stimulated with the stated combinations of cytokines for 3 h, and cells were prepared for flow cytometry analysis detecting IL-17RA, IL-17RC, IL-10Rb, and IL-22Ra1. (B) Cells were stimulated with the stated combinations of cytokines for 5 h, or IL-17A or IL-22 was added 3 h after stimulation with TNF-a, as noted. RNA was extracted and analyzed by RT-qPCR, where IL-17C fold induction (2−DDCt) was controlled against GAPDH mRNA levels. Statistical analysis was completed using a one-way ANOVA with a Dunnett’s multiple comparison test as shown. ****p < 0.0001. Each data point represents three separate experiments of biological replicates.

Article Snippet: Abs used were mouse anti-RelA (1:1000 dilution of F-6, Santa Cruz), mouse anti-p50 (1:500 dilution of E-10, Santa Cruz), mouse anti heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1; 1:5000 dilution of 4B10, Santa Cruz), mouse anti-GAPDH (1:1000 dilution of 6C5, Thermo Fisher Scientific), rabbit anti IL22Ra1 (1:1000 dilution of ab5984, Abcam), goat anti IL-17RA (1:1000 dilution of AF177, R&D Systems), rabbit anti-p38 (1:5000 dilution of D13E1, Cell Signaling Technology), rabbit anti phospho-p38 (1:1000 dilution of 9211, Cell Signaling Technology), rabbit anti-ERK1/2 (1:10,000 dilution of EPR17526, Abcam), rabbit anti phospho-ERK1/2 (1:500 dilution of 197G2, Abcam), rabbit anti-JNK1/2/3 (1:1000 dilution of EPR167979, Abcam), rabbit anti phosphoJNK1/2/3 (1:2000 dilution of EPR5693, Abcam), rabbit anti activating transcription factor (ATF)-2 (1:1000 dilution of D4L2X, Cell Signaling Technology), and rabbit anti phospho-ATF-2 (1:1000 dilution of A8J7P, Cell Signaling Technology).

Techniques: Flow Cytometry, Quantitative RT-PCR, Comparison

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Th17-Derived Cytokines Synergistically Enhance IL-17C Production by the Colonic Epithelium.

doi: 10.4049/jimmunol.2200125

Figure Lengend Snippet: FIGURE 7. Primary non-IBD and UC uninvolved human colonic spheroids, but not UC involved cells, stimulated with Th17-derived cytokines synergistically induce IL-17C expression. (A and B) Primary human colonic spheroids from nonIBD-affected individuals (A) and UC donors (B) were stimulated with TNF-a (50 ng/ml), IL-17A (100 ng/ml), and IL-22 (10 ng/ml). RNA was collected at 5 h, and IL-17C mRNA was quantified as previously described in the legend to Fig. 1. Statistical analysis was completed using a one-way ANOVA with a Dunnett’s multiple comparison test. **p 5 0.0035, ****p < 0.0001. Each data point repre- sents separate experiments of biological replicates for distinct spheroids derived from different donors. (C) Spheroids from non-IBD individuals and UC donors were prepared for flow cytometry analysis detecting IL-17RA, IL-17RC, IL-10Rb, and IL-22Ra1.

Article Snippet: Abs used were mouse anti-RelA (1:1000 dilution of F-6, Santa Cruz), mouse anti-p50 (1:500 dilution of E-10, Santa Cruz), mouse anti heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1; 1:5000 dilution of 4B10, Santa Cruz), mouse anti-GAPDH (1:1000 dilution of 6C5, Thermo Fisher Scientific), rabbit anti IL22Ra1 (1:1000 dilution of ab5984, Abcam), goat anti IL-17RA (1:1000 dilution of AF177, R&D Systems), rabbit anti-p38 (1:5000 dilution of D13E1, Cell Signaling Technology), rabbit anti phospho-p38 (1:1000 dilution of 9211, Cell Signaling Technology), rabbit anti-ERK1/2 (1:10,000 dilution of EPR17526, Abcam), rabbit anti phospho-ERK1/2 (1:500 dilution of 197G2, Abcam), rabbit anti-JNK1/2/3 (1:1000 dilution of EPR167979, Abcam), rabbit anti phosphoJNK1/2/3 (1:2000 dilution of EPR5693, Abcam), rabbit anti activating transcription factor (ATF)-2 (1:1000 dilution of D4L2X, Cell Signaling Technology), and rabbit anti phospho-ATF-2 (1:1000 dilution of A8J7P, Cell Signaling Technology).

Techniques: Derivative Assay, Expressing, Comparison, Flow Cytometry