Fig. 3: Nuclear IL-33 is required for tumor growth and macrophage infiltration.

a Diagram of full-length and mutant IL-33 constructs. Full-length IL-33 consists of a nuclear localization domain (green), chromatin-binding motif (CBM; purple), an activation domain (blue), and an IL-1 like cytokine domain (red). Deletion mutant (ΔNLS) lacks the nuclear domain. IL-33 western blot of U87-IL-33 (clones 3 and 9), ΔNLS (clones 3 and 4), or pcDNA3.1 vector (control) detect full-length IL-33 (33 kDa) in cell lysates and a 25 kDa secreted form in the conditioned medium. Actin was used as a loading control. N = 3 experiments. b Immunofluorescence for nuclear IL-33 (green) in control, IL-33, or ΔNLS transfected U87 cells 24 h after treatment with leptomycin B (50 nM). Scale bar, 20 µm. c Mice implanted with pcDNA (control), IL-33, and ΔNLS-expressing U87 were sacrificed at 3 weeks, when IL-33 mice became symptomatic. Second cohort was sacrificed at 6 weeks postinjection. Shown are representative images of IHC (brown) for h-nucleolin, Iba1, and IL-33. Experiment was repeated five times (N = 5 mice per group/experiment). Sections were counterstained with hematoxylin (blue). Scale bar, 2 mm; inset scale bar, 25 µm. d Kaplan–Meier survival curve of animals bearing U87-control (N = 14), IL-33 (clone 3, N = 10; clone 9, N = 10), or ΔNLS (clone 3, N = 3; clone 4, N = 3) tumors from three independent experiments. The p-value was calculated using log-rank Mantel–Cox test. Control vs. IL-33 clones 3 or 9, ****p ≤ 0.0001; IL-33 clone 9 vs. ΔNLS clone 3, **p = 0.0054. e Quantification of CD11b⁺Gr1⁻ macrophage in control (N = 3) and IL-33 (N = 3) tumors using flow cytometry. PBS-injected mice were used as sham control (N = 4). Graph shows the normalized counts of CD11b⁺Gr1⁻ cells per million total events. Data are mean ± SEM; **p ≤ 0.01, ****p ≤ 0.0001 by one-way ANOVA with Tukey’s post hoc test. f Syngeneic K1492 glioma cells expressing IL-33 or empty vector (control) were allowed to grow for 10 days when IL-33⁺ mice became symptomatic. Representative H&E and Iba1 (brown) IHC images are shown. Experiment was repeated five times (N = 5 per group/experiment). Sections were counterstained with hematoxylin (blue). Scale bar,  1 mm; inset scale bar, 50 µm. g Kaplan–Meier survival curve of animals bearing control (N = 15) or IL-33 (N = 18) tumors from three independent experiments. The p-value was calculated using log-rank Mantel–Cox test (control vs. IL-33, p = 0.0477). See also Supplementary Fig. 3. Source data are available as a Source data file.