human il17br  (Sino Biological)

Journal: American Journal of Cancer Research

Article Title: IL25 + macrophages are a key determinant of treatment resistance of IL17RB + breast cancer

doi:

Figure Lengend Snippet: IL17RB positivity in tumor tissues is a poor prognostic factor in breast cancer. Tumor tissues obtained from stage I-III breast cancer patients (n = 115) were immunohistochemically analyzed for IL17RB expression, and the expression patterns were classified into 3 levels: Level 0, no expression; Level 1, weak expression; and Level 2, strong expression. Diseases-free survival of the patients was statistically compared between two groups divided by the positivity (Level 0 versus Level 1+2).

Article Snippet: These cells were transfected with a plasmid vector pCMV6-ENTRY encoding murine il17rb (NM_019583; Origene) or human il17rb (NM_018725; Origene), or the empty vector as mock control using a polymerase-based transfection reagent jetPEI (Polyplus) according to the manufacturer’s instructions.

Techniques: Expressing

Journal: American Journal of Cancer Research

Article Title: IL25 + macrophages are a key determinant of treatment resistance of IL17RB + breast cancer

doi:

Figure Lengend Snippet: Patient characteristics categorized by IL17RB expression levels in tumors

Article Snippet: These cells were transfected with a plasmid vector pCMV6-ENTRY encoding murine il17rb (NM_019583; Origene) or human il17rb (NM_018725; Origene), or the empty vector as mock control using a polymerase-based transfection reagent jetPEI (Polyplus) according to the manufacturer’s instructions.

Techniques: Expressing

Journal: American Journal of Cancer Research

Article Title: IL25 + macrophages are a key determinant of treatment resistance of IL17RB + breast cancer

doi:

Figure Lengend Snippet: IL17RB transduction confers cancer stem-like properties to murine mammary cancer 4T1 cells. A. IL17RB transduction simultaneously upregulates IL17RA expression, and confers anoikis resistance. Murine mammary cancer 4T1 cells were transfected with a plasmid vector encoding murine il17rb (TR1, TR2, and TR3) or the empty vector as mock control (mock). The immunofluorescence intensity was measured as pixel counts at 400× magnification (3 fields per slide × 2 slides/clone, n = 6). Scale = 100 µm. B. IL17RB transduction confers high self-renewal and invasive on tumor cells (n = 3). Photos show the appearance of sphere colony formation and invaded cells (scale = 100 µm). C. IL17RB transduction simultaneously upregulates gene expression related to cancer stemness. RT-PCR was conducted to analyze gene expression in the tumor cells. D. The IL17RB+ tumor cells are resistant to cyclin-dependent kinase 4/6 inhibitors. The TR cells (closed circles) or mock cells (open circles) were cultured with abemaciclib/LY2835219 (CDKI-LY) or palbociclib/PD0332991 (CDKI-PD) for 2 days, and the cell proliferation was assessed by WST1 assay (n = 3). Graphs were depicted as the percentage of the control without agents (100%). E. CDKI therapy rather promotes IL17RB+ tumor growth in the implanted mice. BALB/c mice were subcutaneously (s.c.) implanted with tumor cells (5 × 105), and were orally administered with CDKI-LY (5 mg/kg) or PBS as a control daily on days 3-7 after tumor implantation (n = 5). Mock + PBS, open circles. Mock + CDKI-LY, closed circles. TR + PBS, open triangles. TR + CDKI-LY, closed triangles. All graphs show means ± SDs. *P < 0.01, **P < 0.05 versus mock control. Representative data of an experiment out of three independent experiments with consistent results.

Article Snippet: These cells were transfected with a plasmid vector pCMV6-ENTRY encoding murine il17rb (NM_019583; Origene) or human il17rb (NM_018725; Origene), or the empty vector as mock control using a polymerase-based transfection reagent jetPEI (Polyplus) according to the manufacturer’s instructions.

Techniques: Transduction, Expressing, Transfection, Plasmid Preparation, Control, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Tumor Implantation

Journal: American Journal of Cancer Research

Article Title: IL25 + macrophages are a key determinant of treatment resistance of IL17RB + breast cancer

doi:

Figure Lengend Snippet: IL17RB+ tumor cells systemically expand IL25+ macrophages in the implanted mice. A. IL17RB+ tumor growth is retarded after implantation in mice. The TR (closed triangles and squares) or mock cells (open circles) were s.c. implanted in mice (5 × 105, n = 5). B. F4/80+ macrophages (Møs) and possibly exhausted CD3+PD1+Tim3+ T cells increase in the TR tumors (day 15 after implantation, n = 3). C. F4/80+ Møs also systemically increase in spleen of the TR-implanted mice (day 15; n = 3). D. Cytotoxic activities of anti-tumor effector cells are extremely reduced in the TR-implanted mice. CD8+ T cells were isolated from spleen cells (SPCs) stimulated with a tumor antigen AH1 peptide (1 µg/ml) for 6 days, and the sorted CD8+ T cells as CTLs were co-cultured with 4T1 cells as a target at ET ratio = 40:1 for 4 hours (n = 3). Splenic DX5+ NK cells were also co-cultured with Yac1 cells as a target at ET ratio = 20:1 for 4 hours (n = 3). E. The IL17RB+ tumor-induced Møs highly produce IL25. F4/80+ Møs were isolated from tumor-infiltrating cells (TILs) and SPCs of mice on day 15 after implantation with mock tumors (mock-Mø) or TR tumors (TR-Mø), and the adherent Møs after 2-hour-culture were stained with anti-IL25 mAb or the isotype control (n = 10, pooled). Scale = 100 µm. The 6-day-cultured splenic Mø supernatant was also tested for IL25 or IL17B by ELISA (n = 3). F. The IL17RB+ tumor-induced Møs suppress CTL induction partly by the secreted IL25. The sorted F4/80+ Møs were cocultured with the AH1-prestimulated CD8+ T cells in the presence of the AH1 peptide (10 µg/ml), IL2 (100 IU/ml), and anti-IL25 mAb or mIgG as a control (10 µg/ml) for 6 days, and the sorted CD8+ T cells were tested for cytotoxic activity (ET ratio = 20:1; n = 3). All graphs show means ± SDs. *P < 0.01, **P < 0.05 versus mock control. Representative data of an experiment out of three independent experiments with consistent results.

Article Snippet: These cells were transfected with a plasmid vector pCMV6-ENTRY encoding murine il17rb (NM_019583; Origene) or human il17rb (NM_018725; Origene), or the empty vector as mock control using a polymerase-based transfection reagent jetPEI (Polyplus) according to the manufacturer’s instructions.

Techniques: Isolation, Cell Culture, Staining, Control, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: American Journal of Cancer Research

Article Title: IL25 + macrophages are a key determinant of treatment resistance of IL17RB + breast cancer

doi:

Figure Lengend Snippet: The IL17RB+ tumor-induced Møs intensify tumor functions. A. The TR-Møs enhance tumor invasive and self-renewal abilities partly utilizing the secreted IL25. Tumor cells were stimulated with IL25 (10 ng/ml) or the 6-day-cultured F4/80+ Mø supernatants (6 days) in the presence of anti-IL25 mAb or mIgG as a control (10 µg/ml) for 3 days before assays (n = 3). B. The TR-Møs promote in vivo tumor progression utilizing the secreted IL25. 4T1 cells were s.c. coinjected (1:1) with splenic Møs in mice, and the subcutaneous tumors were injected with anti-IL25 mAb or mIgG as a control (100 µg) on days 3 and 10 after coinjection (n = 5). Open circles, tumor only. Closed circles, tumor + mock-Møs + mIgG. Gray circles, tumor + mock-Møs + anti-IL25 mAb. Closed triangles, tumor + TR-Møs + mIgG. Gray triangles, tumor + TR-Møs + anti-IL25 mAb. All graphs show means ± SDs. *P < 0.01, **P < 0.05 versus mock. Representative data of an experiment out of three independent experiments with consistent results.

Article Snippet: These cells were transfected with a plasmid vector pCMV6-ENTRY encoding murine il17rb (NM_019583; Origene) or human il17rb (NM_018725; Origene), or the empty vector as mock control using a polymerase-based transfection reagent jetPEI (Polyplus) according to the manufacturer’s instructions.

Techniques: Cell Culture, Control, In Vivo, Injection

Journal: American Journal of Cancer Research

Article Title: IL25 + macrophages are a key determinant of treatment resistance of IL17RB + breast cancer

doi:

Figure Lengend Snippet: Significance of the IL17RB-IL25 axis in human breast cancer system. A. IL17RB transduction confers high self-renewal and invasive properties on human breast cancer (n = 3). Human breast cancer MCF7 cells were transfected with a plasmid vector encoding human il17rb (2 clones: TR1 and TR2) or the empty vector as mock control (mock). Photos show IL17RB expression and anoikis-resistant appearance (scale = 100 µm). B. The IL17RB+ tumor cells are resistant to CDKI treatment. The TR cells (closed triangles and squares) or mock cells (open circles) were cultured with CDKI-LY for 2 days, and the cell proliferation was assessed by WST1 assay (n = 3). Graphs were depicted as the percentage of the control without agents (100%). C. IL17RB+ tumor growth is retarded after implantation in mice. The TR (closed triangles and squares) or mock cells (open circles) were s.c. implanted in immunodeficient nude mice (1 × 106, n = 5). D. The IL17RB+ tumor-expanded Møs highly produce IL25. F4/80+ Møs were isolated from SPCs of the tumor-implanted mice (day 30), and the 6-day-cultured Mø supernatants were tested for IL25 by ELISA (n = 3). E. Combination therapy with CDKI and anti-IL25 mAb significantly suppresses tumor progression in the xenograft models (n = 5). Nude mice were s.c. implanted with parental MCF7 cells (1 × 106), and received two cycles of the treatments: oral administration with CDKI-LY (5 mg/kg) or PBS as a control daily on days 5-9 and days 16-20 after tumor implantation, and/or i.p. injection with anti-IL25 mAb or mIgG as a control (10 mg/kg) on days 5, 9, 16 and 20. Open circles, PBS + mIgG. Closed circles, CDKI-LY + mIgG. Gray triangles, PBS + anti-IL25 mAb. Closed triangles, CDKI-LY + anti-IL25 mAb. All graphs show means ± SDs. *P < 0.01 versus mock. Representative data of an experiment out of three independent experiments with consistent results.

Article Snippet: These cells were transfected with a plasmid vector pCMV6-ENTRY encoding murine il17rb (NM_019583; Origene) or human il17rb (NM_018725; Origene), or the empty vector as mock control using a polymerase-based transfection reagent jetPEI (Polyplus) according to the manufacturer’s instructions.

Techniques: Transduction, Transfection, Plasmid Preparation, Clone Assay, Control, Expressing, Cell Culture, Isolation, Enzyme-linked Immunosorbent Assay, Tumor Implantation, Injection

Journal: mBio

Article Title: Viral Mimicry of Interleukin-17A by SARS-CoV-2 ORF8

doi: 10.1128/mbio.00402-22

Figure Lengend Snippet: ORF8 binding enhances the heterodimerization of hIL-17RA/C. (A) Purified ORF8-Flag interacts with hIL-17RA and hIL-17RC. At 24 h posttransfection of the IL-17 receptor construct, 293T cells were subsequently incubated for 30 min with mock-treated or purified ORF8-Flag, followed by staining with anti-Flag antibody for flow cytometry. (B) Purified ORF8-Flag protein interacts with endogenous IL-17RA and IL-17RC in THP-1 cells. THP-1 cells were incubated with purified ORF8-Flag for 2 h. Cell lysates were immunoprecipitated with anti-Flag, followed by immunoblotting with the indicated antibodies. (C) Effects of IL-17RA/B/C deficiency on the ORF8-induced phosphorylation of p65 and IκBα. IL-17RA, IL-17RB, or IL-17RC knockout (KO) THP-1 cells were generated by the CRISPR-Cas9 method. IL-17RA KO, IL-17RB KO, IL-17RC KO, or control THP-1 cells were mock treated or treated with ORF8 for the indicated times, followed by immunoblotting with the indicated antibodies. (D) Effects of IL-17RA/B/C deficiency on ORF8-mediated cytokine gene expression. IL-17RA KO, IL-17RB KO, IL-17RC KO, or control THP-1 cells were mock treated or treated with ORF8 for 16 h, followed by qRT-PCR to measure the mRNA levels of indicated genes. (E) Proximity ligation assay (PLA) of IL-17RA/C heterodimerization in NIH 3T3 cells. At 24 h posttransfection with HA-hIL-17RA and Myc-hIL-17RC, NIH 3T3 cells were treated with hIL-17A or purified ORF8 for 30 min, followed by PLA. P values were calculated by two-way ANOVA with Dunnett’s posttest in panel D or one-way ANOVA with Tukey’s posttest in panel E. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.0001.

Article Snippet: The pCMV6-IL-17RA and IL-17RB constructs were purchased from Origene.

Techniques: Binding Assay, Purification, Construct, Incubation, Staining, Flow Cytometry, Immunoprecipitation, Western Blot, Knock-Out, Generated, CRISPR, Expressing, Quantitative RT-PCR, Proximity Ligation Assay