recombinant mouse il 36γ  (R&D Systems)

Journal: Basic Research in Cardiology

Article Title: Impact of chronic hyperglycaemia on the coronary microcirculation – benefits of targeting IL-36 and diet reversal

doi: 10.1007/s00395-025-01107-y

Figure Lengend Snippet: Impact of a HFD on cardiac IL-36R/α/β expression and response of coronary microcirculation to IL-36 in vivo. A Representative immunofluorescence images of frozen heart sections to show IL-36R, endothelial cells (ECs; CD31) and oxidative damage expression (not analysed but increased expression in injured HFD mice). B Quantitative analysis of immunofluorescence images for IL-36R expression. N = 4/group. Flow cytometric analysis for IL-36R expression on C ECs and D cardiomyocytes (CMs). N = 3/group. Quantitative analysis of immunofluorescence images (not shown) for E IL-36α and F IL-36β expression. N = 4 mice/group. G Representative intravital images of the healthy beating heart after topical exposure to IL-36γ (100 ng/ml). Images show neutrophils and platelets at 90 min post-treatment. Quantitative analysis of intravital data for adherent H neutrophils and I platelets at 120 min post-exposure to IL-36γ. N = 3/group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 when tested using a one-way ANOVA followed by a Tukey multiple comparison test. ND normal diet; HFD high fat diet. Scale bar: 100 μm

Article Snippet: The ability of IL-36 cytokine to directly mediate neutrophil and/or platelet adhesion was assessed by topically applying recombinant mouse IL-36γ (20 μL at 200 ng/mL; R&D Systems) or PBS for 15 min.

Techniques: Expressing, In Vivo, Immunofluorescence, Comparison

Journal: Frontiers in Immunology

Article Title: Interleukin-36γ is causative for liver damage upon infection with Rift Valley fever virus in type I interferon receptor-deficient mice

doi: 10.3389/fimmu.2023.1194733

Figure Lengend Snippet: IL-36γ is causative for the liver injury in RVFV cl13-infected IFNAR -/- mice. C57BL/6 (WT) and IFNAR -/- mice were i.p. infected with 2x10 4 pfu/200 µl RVFV cl13. (A) IL-36γ was measured 30 hpi within the serum using an ELISA method (n=4-6). (B) C57BL/6 (WT) mice were i.v. injected with 1 µg recombinant IL-36γ. ALT activity within the serum was measured 3 hours post treatment (n=3). (C) RVFV cl13-infected IFNAR -/- mice were i.v injected with 6 µg rIL-36RA in 200 µl 12 and 24 hpi. RVFV cl13-infected IFNAR -/- mice served as control. ALT activity was measured 30 hpi (n=5). (D) Histological analyses were performed using H&E staining. Liver sections of RVFV cl13-infected WT and IFNAR -/- mice were prepared 30 hpi as described earlier . Additionally, RVFV cl13-infected IFNAR -/- mice were rIL-36RA-treated (all n=2). Untreated animals served as controls. Arrows exemplarily indicate apoptotic bodies within the tissue. (E) Organs of WT and IFNAR -/- mice were harvested 30 hours post RVFV infection and analyzed for the viral load by plaque assay (n=2-6). Error bars indicate standard deviations. * < 0.05; ** < 0.01 (Welch’s t-test).

Article Snippet: Recombinant mouse IL-36γ/IL-1F9 (aa 13-164; R&D) was diluted in PBS and intravenously (i.v.) injected (1 μg in a volume of 200 μl).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Injection, Recombinant, Activity Assay, Control, Staining, Plaque Assay

Journal: Frontiers in Immunology

Article Title: Interleukin-36γ is causative for liver damage upon infection with Rift Valley fever virus in type I interferon receptor-deficient mice

doi: 10.3389/fimmu.2023.1194733

Figure Lengend Snippet: PEC are the main source of IL-36γ upon RVFV cl13 infection of IFNAR -/- mice. (A) To exclude genotype-specific differences in basal expression levels, spleen, liver, and peritoneal exudate cells (PEC) of C57BL/6 (WT) and IFNAR -/- mice (each n=4) were isolated and RNA was prepared as described elsewhere . Expression of IL-36γ was determined by qRT-PCR analyses. All values were normalized to WT animals; n.s.=not significant (Mann Whitney test). (B) C57BL/6 (WT) and IFNAR -/- mice (n=4-9) were i.p. infected with 2x10 4 pfu RVFV cl13 in 200 µl. Spleen, liver, and PEC were isolated 24 hpi infection and RNA was prepared as described earlier . Expression of IL-36γ was determined by qRT-PCR analyses. (C) ISRE-eGFP mice were either left untreated or infected with RVFV cl13 for 30 hours. PEC were isolated and analyzed for eGFP expression by flow cytometry. eGFP-positive cells were further characterized as CD11b + F4/80 + (one representative staining out of three is shown). Error bars indicate standard deviations; * < 0.05 (Welch’s t-test); n.s., not significant.

Article Snippet: Recombinant mouse IL-36γ/IL-1F9 (aa 13-164; R&D) was diluted in PBS and intravenously (i.v.) injected (1 μg in a volume of 200 μl).

Techniques: Infection, Expressing, Isolation, Quantitative RT-PCR, MANN-WHITNEY, Flow Cytometry, Staining

Journal: Frontiers in Immunology

Article Title: Interleukin-36γ is causative for liver damage upon infection with Rift Valley fever virus in type I interferon receptor-deficient mice

doi: 10.3389/fimmu.2023.1194733

Figure Lengend Snippet: Myeloid cells need to sense type I IFN in order to protect from IL-36γ-mediated liver injury. IFNAR -/- , Mye-IFNAR -/- , DC-IFNAR -/- , and T-IFNAR -/- mice were i.p. infected with 2x10 4 pfu RVFV cl13 in 200 µl. (A) ALT activity was measured 0 and 30 hpi infection (n=3-4). (B) Induction of IL-36γ within the PEC was determined by qRT-PCR analyses (n=5-8); * < 0.05; ** < 0.01 (Welch’s t-test); n.s., not significant.

Article Snippet: Recombinant mouse IL-36γ/IL-1F9 (aa 13-164; R&D) was diluted in PBS and intravenously (i.v.) injected (1 μg in a volume of 200 μl).

Techniques: Infection, Activity Assay, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Interleukin-36γ is causative for liver damage upon infection with Rift Valley fever virus in type I interferon receptor-deficient mice

doi: 10.3389/fimmu.2023.1194733

Figure Lengend Snippet: MAVS is critically involved in IL-36γ induction and IL-36γ-mediated liver injury upon RVFV cl13 infection of IFNAR -/- mice. IFNAR -/- , MAVS -/- IFNAR -/- , and MyD88 -/- IFNAR -/- mice were i.p. infected with 2x10 4 pfu RVFV cl13 in 200 µl for 30 hours. (A) IL-36γ induction was investigated by qRT-PCR analyses of the PEC (n=4-6). (B) ALT activity was measured in serum samples at 30 hours post infection (n=4-8). (C) Viral load in different organs was analyzed by plaque assay (n=4-7). Error bars indicate standard deviations; * < 0.05; ** < 0.01; n.s., not significant.

Article Snippet: Recombinant mouse IL-36γ/IL-1F9 (aa 13-164; R&D) was diluted in PBS and intravenously (i.v.) injected (1 μg in a volume of 200 μl).

Techniques: Infection, Quantitative RT-PCR, Activity Assay, Plaque Assay

Journal: Cancers

Article Title: Regulation of Interleukin-36γ/IL-36R Signaling Axis by PIN1 in Epithelial Cell Transformation and Breast Tumorigenesis

doi: 10.3390/cancers14153654

Figure Lengend Snippet: Effects of IL-36γ on anchorage-independent growth and epithelial cell transformation in vitro. ( a ) JB6 Cl41 cells were treated with the indicated concentrations of IL-36γ for 48 h, and cell proliferation was determined using the BrdU incorporation assay. ( b , c ) JB6 Cl41 cells were treated with different concentrations of IL-36γ as indicated and grown in a soft agar matrix by incubation at 37 °C in a 5% CO 2 atmosphere for 14 days. Representative images from three separate experiments are shown ( b ), followed by a calculation of the average colony numbers and sizes (diameter > 80 μm) ( c ). ( d ) MCF7 cells were treated with various concentrations of IL-36γ for 48 h, and cell proliferation was determined using the BrdU incorporation assay. ( e , f ) MCF7 cells were treated with the indicated concentrations of IL-36γ and grown in a soft agar matrix by incubation at 37 °C in a 5% CO 2 atmosphere for 14 days. Representative images from three separate experiments are shown ( e ), followed by a calculation of the average colony numbers and sizes (diameter > 100 μm) ( f ). Error bars represent the mean ± standard deviation (S.D.) of triplicate measurements from at least three individual experiments. Statistical analyses were performed using one-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001, compared to the control groups). In b and e, magnification, 100×.

Article Snippet: Recombinant mouse and human IL-36γ were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Transformation Assay, In Vitro, BrdU Incorporation Assay, Incubation, Standard Deviation

Journal: Cancers

Article Title: Regulation of Interleukin-36γ/IL-36R Signaling Axis by PIN1 in Epithelial Cell Transformation and Breast Tumorigenesis

doi: 10.3390/cancers14153654

Figure Lengend Snippet: Effects of IL-36γ on the MEK/ERK and JNK/c-Jun signaling pathway in JB6 Cl41 cells. ( a – d ) Cells were treated with the indicated concentrations of IL-36γ for 30 min ( a , c ) or 10 ng/mL IL-36γ for the indicated times ( b , d ). Proteins in the whole-cell lysates were analyzed by Western blotting. ( e ) Cells were transfected with mouse sgCtrl and sgIL-36R. After 48 h of transfection, cells were serum-starved for 24 h prior to treatment with 10 ng/mL of IL-36γ. After 15 min, total protein was collected, and the levels of the indicated protein were determined by Western blotting using specific antibodies. ( f , g ) Cells were pre-treated with the indicated concentrations of PD98059 ( f ) and SP600125 ( g ) for 12 h and then treated with 10 ng/mL IL-36γ for 15 min. Proteins in the whole-cell lysates were analyzed by Western blotting. ( a – g ) Representative blots are shown from at least three independent experiments. 3.3. PIN1 Enhances AP-1 Activity and IL-36γ-Induced Transformation of JB6 Cl41 Cells. The full uncropped blots for can be found in .

Article Snippet: Recombinant mouse and human IL-36γ were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Transfection, Activity Assay, Transformation Assay

Journal: Cancers

Article Title: Regulation of Interleukin-36γ/IL-36R Signaling Axis by PIN1 in Epithelial Cell Transformation and Breast Tumorigenesis

doi: 10.3390/cancers14153654

Figure Lengend Snippet: Effects of juglone in IL-36γ-induced AP-1 activity and cellular transformation of JB6 Cl41 cells. ( a , b ) Cells were transfected with mock and XP-PIN1 ( a ) or mouse sgCtrl and sgPIN1 ( b ), respectively. After 48 h of transfection, cells were serum-starved for 24 h and treated with 10 ng/mL IL-36γ for 15 min. Proteins in the whole-cell lysates were determined by Western blotting. ( c ) Cells were exposed to the indicated amount of juglone for 12 h, prior to 15 min of treatment with 10 ng/mL IL-36γ. The levels of the indicated proteins were determined by Western blotting using specific antibodies. ( d – f ) The luciferase reporters c-Fos-Luc ( d ), c-Jun-Luc ( e ), and AP-1-Luc ( f ) were co-transfected along with the pRL-TK vector into cells. Cells were serum-starved for 24 h, following 24 h of transfection prior to dose-dependent treatment with IL-36γ for 24 h. ( g ) The luciferase reporter AP-1-Luc was co-transfected with pRL-TK vector into cells. Cells were pre-treated with the indicated doses of juglone, followed by 24 h of serum starvation and then exposed to 10 ng/mL IL-36γ for 24 h. ( h , i ) Cells were treated with 10 ng/mL IL-36γ either alone or in combination with 50 μM juglone in a soft agar matrix and incubated at 37 °C in a 5% CO 2 atmosphere for 14 days. Representative pictures of the colonies from three separate experiments ( h ) and the quantification of the colony sizes and numbers ( i ). Magnification, 100×. Error bars indicate the mean ± S.D. of triplicate measurements. Statistical analyses were performed using one-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001, compared to the control group or only-IL-36γ-treated group). The full uncropped blots for can be found in .

Article Snippet: Recombinant mouse and human IL-36γ were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Activity Assay, Transformation Assay, Transfection, Western Blot, Luciferase, Plasmid Preparation, Incubation

Journal: Cancers

Article Title: Regulation of Interleukin-36γ/IL-36R Signaling Axis by PIN1 in Epithelial Cell Transformation and Breast Tumorigenesis

doi: 10.3390/cancers14153654

Figure Lengend Snippet: Role of PIN1 in the IL-36γ-induced MEK/ERK and JNK/c-Jun signaling pathways in MCF7 cells. ( a , b ) Cells were treated with the indicated concentration of IL-36γ for 30 min ( a ) or treated with 10 ng/mL IL-36γ for the indicated time ( b ). ( c ) Cells were transfected with human sgCtrl and sgIL-36R. After 48 h, cells were serum-starved for 24 h prior to treatment with 10 ng/mL IL-36γ. After 15 min, cells were harvested and lysed. Proteins in the whole-cell lysate were determined by Western blotting. ( d , e ) Cells were transfected with mock and XP-PIN1 ( d ) or sgCtrl and sgPIN1 ( e ), respectively. At 48 h after transfection, cells were serum-starved for 24 h and treated with 10 ng/mL IL-36γ for 15 min. ( f ) Cells were pre-treated with the indicated doses of juglone for 12 h prior to treatment with 10 ng/mL IL-36γ. After 15 min, cells were harvested and lysed. Proteins in the whole-cell lysates were analyzed by Western blotting. The blots shown are representative of at least three independent experiments.The full uncropped blots for can be found in .

Article Snippet: Recombinant mouse and human IL-36γ were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Concentration Assay, Transfection, Western Blot

Journal: Cancers

Article Title: Regulation of Interleukin-36γ/IL-36R Signaling Axis by PIN1 in Epithelial Cell Transformation and Breast Tumorigenesis

doi: 10.3390/cancers14153654

Figure Lengend Snippet: Role of PIN1 in IL-36γ-induced AP-1 activity and epithelial cell transformation. ( a – c ) The luciferase reporters c-Fos-Luc ( a ), c-Jun-Luc ( b ), and AP-1-Luc ( c ) were co-transfected with the pRL-TK vector into cells. Following 24 h of transfection, cells were serum-starved for 24 h prior to dose-dependent treatment with IL-36γ for 24 h. ( d ) The luciferase reporter AP-1-Luc and sgCtrl or AP-1-Luc and sgPIN1 were co-transfected into cells and incubated for 24 h. After being serum-starved for 24 h, cells were exposed to 10 ng/mL IL-36γ for another 24 h. ( e ) sgCtrl and sgPIN1 were transfected into cells and incubated for 48 h. Then, cells were treated with or without 10 ng/mL IL-36γ in a soft agar matrix and incubated at 37 °C in a 5% CO 2 atmosphere for 14 days. Magnification, 100×. ( f ) Representative images from three separate experiments are presented (left), followed by a calculation of the average colony numbers and sizes (diameter > 200 μm, right). Error bars indicate the mean ± S.D. of triplicate measurements from three individual experiments. Statistical analyses were performed using one-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001, compared to the control group or only-IL-36γ-treated group).

Article Snippet: Recombinant mouse and human IL-36γ were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Activity Assay, Transformation Assay, Luciferase, Transfection, Plasmid Preparation, Incubation

Journal: Cancers

Article Title: Regulation of Interleukin-36γ/IL-36R Signaling Axis by PIN1 in Epithelial Cell Transformation and Breast Tumorigenesis

doi: 10.3390/cancers14153654

Figure Lengend Snippet: Role of PIN1 in IL-36γ-induced cell proliferation and mammary gland tumorigenesis. ( a ) 4T1 cells were seeded and treated with IL-36γ as indicated. Cell proliferation was estimated using the BrdU incorporation assay. ( b , c ) 4T1 cells were treated with the indicated doses of IL-36γ, subjected to a soft agar matrix, and incubated at 37 °C in a 5% CO 2 atmosphere for 14 days. Representative images from three separate experiments are presented ( b ), followed by a calculation of the average colony numbers and sizes (diameter >200 μm) ( c ). ( d , e ) 4T1 cells were treated with the indicated concentrations of IL-36γ in the presence or absence of juglone, grown in a soft agar matrix, and incubated at 37 °C in a 5% CO 2 atmosphere for 14 days. Representative images from three separate experiments are presented ( d ), followed by a calculation of the average colony numbers and sizes (diameter > 200 μm) ( e ). ( f , g ) WT 4T1 and PIN1 KO 4T1 cells were injected into the mammary gland of BALB/c mice in the presence or absence of 100 ng/mL IL-36γ and allowed to grow until tumors were formed. Shown are representative images of tumor ( f ), measured tumor weights, and tumor volumes ( g ). Error bars indicate the mean ± S.D. of triplicate measurements from three independent experiments. Statistical analyses were performed using one-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001, compared to the control group or only-IL-36γ-treated group, respectively). In b and d, magnification, 100×.

Article Snippet: Recombinant mouse and human IL-36γ were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: BrdU Incorporation Assay, Incubation, Injection