Journal: Journal of Neuroscience

Article Title: System xc Activity and Astrocytes Are Necessary for Interleukin-1 -Mediated Hypoxic Neuronal Injury

doi: 10.1523/jneurosci.2459-07.2007

Figure Lengend Snippet: Figure 2. Role of IL-1RI signaling in the potentiation of hypoxic neuronal injury by IL-1 in vitro. A, B, Mixed cortical cell cultures were treated with 1 ng/ml IL-1 for 20–24 h in the presenceorabsenceofrIL-1ra(10–1000ng/ml;A)oranti-IL-1RI(0.1–100g/ml;B),washed, andthendeprivedofoxygen(5h).Thepercentageoftotalneuronalcelldeathwasdetermined 20–24hlater.Anasteriskindicatesvaluessignificantlygreaterthanhypoxiaalone,whereas# denotes a significant diminution of the IL-1-mediated increase in injury (IL-1) as deter- mined by one-way ANOVA followed by a Student–Newman–Keuls t test. Significance was assessed at p 0.05 (n 3–9 cultures pooled from 2–3 different experiments).

Article Snippet: In experiments using recombinant mouse IL-1ra (rIL-1ra) (R & D Systems) or monoclonal anti-mouse IL-1RI antibody directed against the extracellular domain of mouse IL-1RI (MAB7711; R & D Systems), the respective solutions were prepared with IL-1 (1.5 ng/ml; 1.5 ) and added as described above.

Techniques: In Vitro

Journal: Scientific Reports

Article Title: T. gondii infection induces IL-1R dependent chronic cachexia and perivascular fibrosis in the liver and skeletal muscle

doi: 10.1038/s41598-020-72767-0

Figure Lengend Snippet: Chronic Toxoplasma infection causes sustained cachexia in mice. 10–14 week old C57BL/6J mice were intraperitoneally infected with 10 Me49-GFP-luciferase T. gondii cysts (I, red) or mock injected with PBS (UI, black). ( a ) Schematic of weight loss relative to parasite distribution. The acute phase of infection (white) is dominated by Toxoplasma tachyzoites (green crescents) which spread systemically, infecting most tissues in the body. 4–6 weeks post-infection (wpi), systemic infection is largely cleared and parasites are driven to the chronic tissue cyst form (green circles). ( b ) Mice lose up to 20% of their initial body mass in the first 4 wpi and fail to regain weight relative to uninfected controls. N = 35–45 mice pooled from 3 independent experiments. ( c ) Dolichos biflorous positive T. gondii cysts per half brain at 5–9 wpi. N = 19 pooled from 6 independent experiments. ( d ) Daily food intake per cage normalized to pooled weight of the mice in the cage measured every 24 h. N = 7–9 cages per group, pooled from 3 independent experiments. ( e ) Mice were individually housed for 24 h at 10 wpi and food intake over 24 h was determined by weight (left) and caloric content of fecal pellets were determined by bomb calorimetry (right). N = 4 mice per group. ( f ) Echo MRI quantification of fat (left) and lean (right) tissue mass at 2 or 6 wpi. N = 28–45 mice per group, representative of 3 independent experiments. ( g ) Inguinal subcutaneous white adipose tissue (scWAT), epididymal visceral white adipose tissue (vWAT), quadriceps (Quad) and liver weights at 2 wpi or 9 wpi. N = 12–18 mice per group, pooled from 3 experiments. ( h ) Quantity of T. gondii DNA relative to host beta-actin in the tibialis anterior muscle at 9 wpi. N = 4–5 mice per group. n.d. not detectable. ( i ) Serum cytokines measured by Luminex at 1 or 5 wpi. N = 3–4 mice per group, representative of 2 experiments. Error bars are standard error of the mean. *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001 by unpaired Student’s t test.

Article Snippet: 1 × 10 4 cells were seeded overnight onto poly-D-lysine coated glass coverslips in 24-well plates and then stimulated with 10 ng/mL recombinant mouse IL-1α (R&D), 10 ng/mL recombinant mouse TGF-β (R&D), or media alone for 48 h. Coverslips were fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 for 15 min, blocked in 1% BSA for 30 min, and then stained overnight at 4 °C with anti α-SMA antibody (Invitrogen, clone IA4).

Techniques: Infection, Luciferase, Injection, Luminex

Journal: Scientific Reports

Article Title: T. gondii infection induces IL-1R dependent chronic cachexia and perivascular fibrosis in the liver and skeletal muscle

doi: 10.1038/s41598-020-72767-0

Figure Lengend Snippet: Cells expressing IL-1⍺ and IL-1R are observed in the fibrotic liver environment. ( a ) Cytokines in tissue lysates from mice at 9 wpi were measured by ELISA in liver. Data are presented as fold change relative to the mean of uninfected levels. N = 11–12 mice per group, pooled from three independent experiments. ( b ) IL-1α levels in the sera at 9 wpi measured by ELISA. N = 9–14 mice per group, pooled from four independent experiments. ( c ) Immunofluorescence labeling of nuclei (DAPI white), IL-1⍺ (green), CD45 (red), and collagen1⍺1 (blue) in the liver of UI or 9 wpi WT mice . Number of cells staining positive for CD45 and/or IL-1⍺, average 2–3 fields of view where immune infiltrate was present from 3 mice per condition are quantified on the right. Error bars are standard deviation. ( d ) Immunofluorescent labeling of nuclei (DAPI white), ⍺-smooth muscle actin (green), IL-1R (red), and collagen1⍺ (blue) in the liver of uninfected or 9 wpi WT. Inset, arrow head represents ⍺-smooth muscle actin/IL-1R co-staining cells (arrow heads). Number of cells staining positive for IL-1R and/or ⍺-SMA, average of 4–8 fields of view from 3 mice are quantified on the right. Error bars are standard deviation. ( c , d ) represent maximum intensity projections of 9–13 μm thick z-stacks. Scale bar represents 50 μm. Error bars are standard error of the mean except where noted otherwise. *P < 0.05; **P < 0.01; ***P < 0.001 by unpaired Student’s t test.

Article Snippet: 1 × 10 4 cells were seeded overnight onto poly-D-lysine coated glass coverslips in 24-well plates and then stimulated with 10 ng/mL recombinant mouse IL-1α (R&D), 10 ng/mL recombinant mouse TGF-β (R&D), or media alone for 48 h. Coverslips were fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 for 15 min, blocked in 1% BSA for 30 min, and then stained overnight at 4 °C with anti α-SMA antibody (Invitrogen, clone IA4).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Labeling, Staining, Standard Deviation

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

Article Title: Retargeting Interleukin-2 Signaling to NKG2D-Expressing Tumor Infiltrating Leukocytes Improves Adoptive Transfer Immunotherapy

doi: 10.4049/jimmunol.2000926

Figure Lengend Snippet: CD25 (IL-2Rα) and NKG2D were immobilized to individual flow cells of a sensor chip by primary amine chemistry. Serial dilutions of IL-2, mutIL-2, and OMCPmutIL-2 were injected across each flow cell and association and dissociation phases were measured. Renditioned graphic model of protein binding (left) with experimental binding to CD25 (middle) and NKG2D (right) for wild type human IL-2 (A), mutant human (R38A, F42K) IL-2 (B), and OMCPmutIL-2 (C). Experimental binding curves are shown for a representative experiment. (D) For binding of CD25:wild-type IL-2 and OMCPmutIL-2:NKG2D experimental curves were fitted using ANABEL software and the observed binding constant (Kobs) was measured for each concentration. The plot of Kobs vs concentration is shown with the slope as the association rate (Kon) and y-axis intercept as the dissociation rate (Koff).

Article Snippet: Surface plasmon resonance (SPR) A ProteOn XPR36 instrument (BioRad) was used to determine the kinetics of interaction of WT IL-2, mutIL-2, and OMCPmutIL-2 for murine CD25 (#2438-RM, R&D systems) and murine NKG2D-Fc (#139-NK-050, R&D systems).

Techniques: Injection, Protein Binding, Binding Assay, Mutagenesis, Software, Concentration Assay

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

Article Title: Retargeting Interleukin-2 Signaling to NKG2D-Expressing Tumor Infiltrating Leukocytes Improves Adoptive Transfer Immunotherapy

doi: 10.4049/jimmunol.2000926

Figure Lengend Snippet: (A) Fold TIL expansion of murine B16ova -resident leucocytes. (B) Surface expression of NKG2D and IL-2R alpha chain on mouse antigen specific CD8+ T cells compared to those with ovalbumin non-reactive TCR. (C) Fold TIL expansion of Human melanoma -derived TILS in either wild-type IL-2 (blue) or OMCPmutIL-2 (red) (C). *p<.05; **p<.01, ***p<.001, NS p>.05

Article Snippet: Surface plasmon resonance (SPR) A ProteOn XPR36 instrument (BioRad) was used to determine the kinetics of interaction of WT IL-2, mutIL-2, and OMCPmutIL-2 for murine CD25 (#2438-RM, R&D systems) and murine NKG2D-Fc (#139-NK-050, R&D systems).

Techniques: Expressing, Derivative Assay

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

Article Title: Retargeting Interleukin-2 Signaling to NKG2D-Expressing Tumor Infiltrating Leukocytes Improves Adoptive Transfer Immunotherapy

doi: 10.4049/jimmunol.2000926

Figure Lengend Snippet: (A) Number of TIL leucocytes transferred into CD45.1 congenic tumor-bearing recipients after expansion in either wild-type IL-2 or OMCPmutIL-2. Flow cytometric analysis performed 24 hours after transfer. (B)Immunohistochemical analysis of B16ova tumors in wild-type C57Bl/6 mice injected with 20×106 TILs from C57Bl/6EGFP mice expanded for 2 weeks in either wild-type IL-2 or OMCPmutIL-2. Histologic evaluation performed 120 hours after transfer. (C) Relative expression, by median fluorescence intensity, of LFA-1, CD49a or CXCR3 on TIL-resident CD8+ T cells, NK cells or gamma delta T cells after 2-week expansion in IL-2 (blue) or OMCPmutIL-2 (red). (D) Relative expression, by median fluorescence intensity, of LFA-1 on TIL-resident CD8+ T cells, NK cells or γδ T cells after either MDSC or Treg depletion prior to expansion in IL-2 (blue) or OMCPmutIL-2 (red). *p<.05; **p<.01, ***p<.001, NS p>.05

Article Snippet: Surface plasmon resonance (SPR) A ProteOn XPR36 instrument (BioRad) was used to determine the kinetics of interaction of WT IL-2, mutIL-2, and OMCPmutIL-2 for murine CD25 (#2438-RM, R&D systems) and murine NKG2D-Fc (#139-NK-050, R&D systems).

Techniques: Immunohistochemical staining, Injection, Expressing, Fluorescence

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

Article Title: Retargeting Interleukin-2 Signaling to NKG2D-Expressing Tumor Infiltrating Leukocytes Improves Adoptive Transfer Immunotherapy

doi: 10.4049/jimmunol.2000926

Figure Lengend Snippet: (A) NKG2D expression in CD8+ TILs. (B) Expansion of NKG2Dlow or NKG2Dhigh CD8+ T cells in OMCPmutIL-2 or wild-type IL-2. (C) Expression of NKG2D (top), perforin (middle), and FasLigand (bottom) on flow cytometrically sorted NKG2Dlow CD8+ T cells immediately post sort (black line bar open circles), after 7 days of culture in wild-type IL-2 (blue bar, open circles) or OMCPmutIL-2 (red bar, open circles). ***p<.001

Article Snippet: Surface plasmon resonance (SPR) A ProteOn XPR36 instrument (BioRad) was used to determine the kinetics of interaction of WT IL-2, mutIL-2, and OMCPmutIL-2 for murine CD25 (#2438-RM, R&D systems) and murine NKG2D-Fc (#139-NK-050, R&D systems).

Techniques: Expressing

Journal: Journal of Veterinary Science

Article Title: Inhibitory effects of interleukin-10 plasmid DNA on the development of atopic dermatitis-like skin lesions in NC/Nga mice

doi: 10.4142/jvs.2010.11.3.213

Figure Lengend Snippet: Induction of dermatitis and interleukin (IL)-10 plasmid DNA injection schedule. Dorsal regions of mice were shaved on day 0. The hairless dorsal regions and glabrous ears of mice were sensitized with 200 µL of 1% (w/v) DNCB solution on day 4. Three days after sensitization, the dorsal skin and ears were challenged with 150 µL of 0.2% (w/v) DNCB solution every 3 days. Following in vivo delivery of IL-10 plasmid DNA, mice were intradermally injected with 100 µg of IL-10 plasmid DNA in 100 µL of phosphate-buffered saline (PBS) or PBS alone (control) on days 1 and 8 of experiment.

Article Snippet: Forty-eight hours after passage, the cell culture fluids were collected and analyzed for expression of mouse IL-10 protein by the mouse IL-10 Quantikine ELISA Kit (R&D Systems, USA) according to the manufacturer's instructions.

Techniques: Plasmid Preparation, Injection, In Vivo, Saline

Journal: Journal of Veterinary Science

Article Title: Inhibitory effects of interleukin-10 plasmid DNA on the development of atopic dermatitis-like skin lesions in NC/Nga mice

doi: 10.4142/jvs.2010.11.3.213

Figure Lengend Snippet: Clinical skin severity score in the control mice increased gradually with the number of cutaneous applications of DNCB and reached a peak at the end of experiment. A significant improvement in mice injected with IL-10 plasmid DNA had occurred at day 13 ( * p <0.05) and by day 16 was even more pronounced ( ** p <0.01) compared with the control mice (mean ± SD; n = 5).

Article Snippet: Forty-eight hours after passage, the cell culture fluids were collected and analyzed for expression of mouse IL-10 protein by the mouse IL-10 Quantikine ELISA Kit (R&D Systems, USA) according to the manufacturer's instructions.

Techniques: Injection, Plasmid Preparation

Journal: Journal of Veterinary Science

Article Title: Inhibitory effects of interleukin-10 plasmid DNA on the development of atopic dermatitis-like skin lesions in NC/Nga mice

doi: 10.4142/jvs.2010.11.3.213

Figure Lengend Snippet: The skin features demonstrated visually a marked reduction in severity of the dermatitis and more rapid hair re-growth with IL-10 plasmid DNA injection. (A) Dermatitis induced and PBS injected mice, (B) IL-10 plasmid DNA injected mice after dermatitis induction.

Article Snippet: Forty-eight hours after passage, the cell culture fluids were collected and analyzed for expression of mouse IL-10 protein by the mouse IL-10 Quantikine ELISA Kit (R&D Systems, USA) according to the manufacturer's instructions.

Techniques: Plasmid Preparation, Injection

Journal: Journal of Veterinary Science

Article Title: Inhibitory effects of interleukin-10 plasmid DNA on the development of atopic dermatitis-like skin lesions in NC/Nga mice

doi: 10.4142/jvs.2010.11.3.213

Figure Lengend Snippet: The inhibitory effects of interleukin-10 plasmid DNA. The concentration of serum IL-10 in mice injected with IL-10 plasmid DNA was significantly higher than in control mice. * p < 0.05 vs . control, ** p < 0.01 vs . control.

Article Snippet: Forty-eight hours after passage, the cell culture fluids were collected and analyzed for expression of mouse IL-10 protein by the mouse IL-10 Quantikine ELISA Kit (R&D Systems, USA) according to the manufacturer's instructions.

Techniques: Plasmid Preparation, Concentration Assay, Injection

Journal: Journal for Immunotherapy of Cancer

Article Title: CD25-targeted antibody–drug conjugate depletes regulatory T cells and eliminates established syngeneic tumors via antitumor immunity

doi: 10.1136/jitc-2020-000860

Figure Lengend Snippet: Structure and in vitro characterization of CD25-ADC. (A) Structure and (B) in vitro characterization of CD25-ADC. (i) ELISA showing binding of anti-CD25 antibody PC61 to mouse recombinant CD25. (ii–iv) Flow cytometry measurement of PC61 and isotype-control antibody binding to Yac-1, MC38 and CT26 cells. (v–vii) Yac-1, MC38 and CT26 cells’ viability after exposure to CD25-ADC and isotype-ADC (and the naked pyrrolobenzodiazepine-dimer SG3199 in MC38 and CT26 cell lines). MFI, median fluorescence intensity; PABA, para amino benzoic acid.

Article Snippet: Binding of PC61 to mouse recombinant CD25 (R&D Systems) was determined by ELISA, using a mouse CD25/human Fc chimeric antigen (R&D Systems) and a secondary goat antirat HRP (Jackson Immunoresearch Laboratories).

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Binding Assay, Recombinant, Flow Cytometry, Control, Fluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: CD25-targeted antibody–drug conjugate depletes regulatory T cells and eliminates established syngeneic tumors via antitumor immunity

doi: 10.1136/jitc-2020-000860

Figure Lengend Snippet: In vivo antitumor activity of CD25-ADC in the s.c. MC38 syngeneic model. Treatment with (i) vehicle, (ii) anti-PD-1 antibody (5 mg/kg, on days 2, 5, and 8), (iii) non-binding ADC (1 mg/kg, single dose on day 1) alone or (iv) in combination with anti-PD-1 antibody, (v–vii) CD25-ADC (0.1, 0.5, and 1 mg/kg single dose on day 1) alone or (viii–x) in combination with anti-PD-1 antibody, started at a group mean tumor volume of 103 mm 3 . Data are shown as tumor volumes (mm 3 ) over time for each individual mouse (10 mice/group). (xi) Survival of mice shown in i–x. Lines for G4, G5, G8 and G9 are overlapping.

Article Snippet: Binding of PC61 to mouse recombinant CD25 (R&D Systems) was determined by ELISA, using a mouse CD25/human Fc chimeric antigen (R&D Systems) and a secondary goat antirat HRP (Jackson Immunoresearch Laboratories).

Techniques: In Vivo, Activity Assay, Binding Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: CD25-targeted antibody–drug conjugate depletes regulatory T cells and eliminates established syngeneic tumors via antitumor immunity

doi: 10.1136/jitc-2020-000860

Figure Lengend Snippet: In vivo antitumor activity of CD25-ADC in the s.c. CT26 syngeneic model. Treatment with (i) vehicle, (ii) anti-PD-1 antibody (5 mg/kg, on days 2, 5, and 8), (iii) isotype-ADC (1 mg/kg, single dose on day 1) alone or (iv) in combination with anti-PD-1 antibody, (v–vii) CD25-ADC (0.1, 0.5, and 1 mg/kg single dose on day 1) alone or (viii–x) in combination with anti-PD-1 antibody, started at a group mean tumor volume of 110 mm 3 . Data are shown as tumor volumes (mm 3 ) over time for each individual mouse (10 mice/group). (xi) Survival of mice shown in i–x.

Article Snippet: Binding of PC61 to mouse recombinant CD25 (R&D Systems) was determined by ELISA, using a mouse CD25/human Fc chimeric antigen (R&D Systems) and a secondary goat antirat HRP (Jackson Immunoresearch Laboratories).

Techniques: In Vivo, Activity Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: CD25-targeted antibody–drug conjugate depletes regulatory T cells and eliminates established syngeneic tumors via antitumor immunity

doi: 10.1136/jitc-2020-000860

Figure Lengend Snippet: Role of CD8+ T eff cells in CD25-ADC antitumor activity in the MC38 syngeneic model. Depletion of CD8+ T eff cells significantly reduces the antitumor activity of CD25-ADC. CD25-ADC was administered intraperitoneally (i.p.) at a group mean tumor volume of 89 mm 3 as a single dose on day 1 at 0.5 mg/kg alone or in combination with anti-PD-1 antibody (5 mg/kg, on days 2, 5, and 8). Anti-CD8 T-cell depleting antibody (10 mg/kg) was injected i.p. on days 0, 5, 8, and 13. Data are shown as mean tumor volumes (mm 3 ) ± SEM over time (n=10/group).

Article Snippet: Binding of PC61 to mouse recombinant CD25 (R&D Systems) was determined by ELISA, using a mouse CD25/human Fc chimeric antigen (R&D Systems) and a secondary goat antirat HRP (Jackson Immunoresearch Laboratories).

Techniques: Activity Assay, Injection

Journal: Journal for Immunotherapy of Cancer

Article Title: CD25-targeted antibody–drug conjugate depletes regulatory T cells and eliminates established syngeneic tumors via antitumor immunity

doi: 10.1136/jitc-2020-000860

Figure Lengend Snippet: Intratumoral T-cell immunophenotype analysis in MC38-bearing mice. (A) Absolute quantification of intratumoral T regs , CD8+ T cells and CD8+/T reg ratio following i.p. treatment with anti-PD-1 antibody or CD25-ADC or the combination of CD25-ADC and anti-PD-1. (B) Percentage of CD69+, Ki67+ and IFNγ+ tumor-infiltrating CD8+ T cells. Tumors were processed at the indicated times (days post CD25-ADC dose). Horizontal bars represent median value. Statistical differences between treatment groups were calculated using JMP 15 by the Dunn method for joint ranking. Results were considered significant when p<0.05. *, p≤0.05; **, p≤0.01. IFN, interferon.

Article Snippet: Binding of PC61 to mouse recombinant CD25 (R&D Systems) was determined by ELISA, using a mouse CD25/human Fc chimeric antigen (R&D Systems) and a secondary goat antirat HRP (Jackson Immunoresearch Laboratories).

Techniques: Quantitative Proteomics

Journal: Journal for Immunotherapy of Cancer

Article Title: CD25-targeted antibody–drug conjugate depletes regulatory T cells and eliminates established syngeneic tumors via antitumor immunity

doi: 10.1136/jitc-2020-000860

Figure Lengend Snippet: Circulating and thymic T-cell immunophenotype analysis in MC38-bearing mice. (A) Absolute quantification of circulating T regs , CD8+ T cells and CD8+/T reg ratio following i.p. treatment with anti-PD-1 antibody or CD25-ADC or the combination of CD25-ADC and anti-PD-1. Blood was processed at the indicated times (days post CD25-ADC dose). (B) Absolute quantification of thymic T reg cells and CD8+/T reg ratio following i.p. treatment with anti-PD-1 antibody or CD25-ADC or the combination of CD25-ADC and anti-PD-1. Thymus was processed at the indicated times (days post CD25-ADC dose). Statistical differences between treatment groups were calculated using JMP 15 by the Dunn method for joint ranking. Results were considered significant when p<0.05. *, p≤0.05; **, p≤0.01.

Article Snippet: Binding of PC61 to mouse recombinant CD25 (R&D Systems) was determined by ELISA, using a mouse CD25/human Fc chimeric antigen (R&D Systems) and a secondary goat antirat HRP (Jackson Immunoresearch Laboratories).

Techniques: Quantitative Proteomics

Journal: Journal for Immunotherapy of Cancer

Article Title: CD25-targeted antibody–drug conjugate depletes regulatory T cells and eliminates established syngeneic tumors via antitumor immunity

doi: 10.1136/jitc-2020-000860

Figure Lengend Snippet: T-cell dynamic study in non-tumor-bearing mice. Effect of CD25-ADC on the percentage of T regs and T eff levels in non-tumor-bearing mice. Female C57BL/6 mice were injected i.p. with vehicle, CD25-ADC (0.5 mg/kg), or isotype control ADC (0.5 mg/kg) on day 0. (A) Spleen, (B) lymph node, and (C) thymus were collected 4 hours post dose, and 6, 13, and 20 days post dose for T-cell immune profiling. Levels of T regs , CD8+ T, and conventional CD4+ T cells in spleen, lymph nodes, and thymus are presented as % of CD45 cells±SEM over time.

Article Snippet: Binding of PC61 to mouse recombinant CD25 (R&D Systems) was determined by ELISA, using a mouse CD25/human Fc chimeric antigen (R&D Systems) and a secondary goat antirat HRP (Jackson Immunoresearch Laboratories).

Techniques: Injection, Control

Journal: Communications Biology

Article Title: Inflammation impacts androgen receptor signaling in basal prostate stem cells through interleukin 1 receptor antagonist

doi: 10.1038/s42003-024-07071-y

Figure Lengend Snippet: A Representative H&E images of naïve organoids treated with recombinant IL-1RA or neutralizing antibody (Ab) against IL-1α, showing an increase in stratification of treated organoids (Scale bar, 50 μm). B Organoid formation with naïve bPSC was significantly increased with recombinant IL-1RA (50 ng/mL) or IL-1α neutralizing Ab treatment (5 μg/mL) (Naïve_control, n = 12; Naïve_IL-1RA, n = 12; Naïve_IgG, n = 6; Naïve_IL-1α Ab, n = 9). C Quantitation of AR+ (both overall and nuclear) cells in naïve or inflamed organoids treated with IL-1RA (50 ng/mL) (All groups, n = 5). D AR staining (overall and nuclear) was increased in naïve organoids treated with an IL-1α neutralizing Ab (5 μg/mL) and decreased in inflamed organoids treated with an IL-1RA neutralizing Ab (5 μg/mL) (All groups, n = 5). E qRT-PCR showing that the AR target gene Steap4 , which was induced in inflamed organoids and suppressed with Enz (left, n = 3), was upregulated with IL-1RA in naïve organoids and Enz treatment abolished the upregulation (right, n = 3). F The effects of IL-1RA or IL-1α neutralizing Ab were lost with naïve organoids derived from Ar_ flox/y bPSC which were deprived of AR (All groups, n = 6). Data presented in this figure are mean ± SEM.

Article Snippet: The following treatments were used with vehicle controls: Enzalutamide (Selleckchem), R1881 (Sigma-Aldrich), recombinant mouse IL-1RA (R&D Systems), recombinant mouse IL-1α (R&D), IL1-RA neutralizing antibody (#AF-480-NA, R&D) and IL-1α neutralizing antibody (#AF-400-NA, R&D).

Techniques: Recombinant, Control, Quantitation Assay, Staining, Quantitative RT-PCR, Derivative Assay

Journal: Communications Biology

Article Title: Inflammation impacts androgen receptor signaling in basal prostate stem cells through interleukin 1 receptor antagonist

doi: 10.1038/s42003-024-07071-y

Figure Lengend Snippet: A Il1rn and Il1a (arrows) are among the most upregulated genes in the inflamed bPSC compared to naïve bPSC as analyzed with scRNA-seq. B Violin plots of Il1rn, Il1a and Il1r1 expression in naïve and inflamed bPSC. C qRT-PCR of Il1rn isoforms and Il1a in naïve and inflamed bPSC (Naïve, n = 3–4; Inflamed, n = 3–8). D Upregulation of IL-1RA protein is confirmed using ELISA with lysates collected from naïve or inflamed bPSC (Naïve or Inflamed, n = 4). Data presented in this figure are mean ± SEM.

Article Snippet: The following treatments were used with vehicle controls: Enzalutamide (Selleckchem), R1881 (Sigma-Aldrich), recombinant mouse IL-1RA (R&D Systems), recombinant mouse IL-1α (R&D), IL1-RA neutralizing antibody (#AF-480-NA, R&D) and IL-1α neutralizing antibody (#AF-400-NA, R&D).

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay