Fig 2: Adhesion to VCAM-1/ICAM-1 up-regulates mRNA expression of inflammatory cytokines. A Stimulation (3 h) with immobilized (orange) but not with soluble (dark grey) VCAM-1/ICAM-1 induces mRNA expression of Il1a, Il1b, Il6, Tnf and Cxcl10 in 32D JAK2-WT (left; n = 3) and 32D JAK2-VF (right; n = 3) cells in comparison to BSA (light grey). Data are shown as fold change (2−ΔΔCT) with mean + SEM. 2-Way ANOVA with Tukey’s multiple comparison test P* < 0.05, P** < 0.01, P*** < 0.001. Cartoon depicting the experimental design, created with Biorender.com (https://BioRender.com/d34c738). B Il1a and Il1b mRNA expression upon VCAM-1/ICAM-1 stimulation (1 h) using isolated granulocytes of Vav1-Cre x Jak2+/+ (n = 7) and Vav1-Cre x Jak2VF/+ mice (n = 7) is shown as fold change compared to the BSA control (“unstimulated”). Data are shown as fold change (2−ΔΔCT) with mean + SEM. RM one-way ANOVA (Šídák's multiple comparisons test) with P* < 0.05, P*** < 0.001. C Stimulation with immobilized VCAM-1/ICAM-1 induces mRNA expression of Il1a, Il1b, Il6, Tnf and Cxcl10 in total bone marrow cells (BMC) of Vav1-Cre x Jak2+/+ (n = 3) and Vav1-Cre x Jak2VF/+ mice (n = 2) in comparison to BSA (grey columns). Data are shown as fold change (2−ΔΔCT) with mean + SEM. D Upon adhesion to VCAM-1/ICAM-1, the formation of β1-integrin clusters in 32D JAK2-WT (blue; n = 144 cells) and 32D JAK2-VF cells (red; n = 96 cells) is measured by fluorescence microscopy (Created in BioRender. Haage, R. (2025) https://BioRender.com/e52r401). The clusters per cell (upper middle) and surface area per cell in pixel (upper right) are increased in the 32D JAK2-VF cells after adhesion on immobilized VCAM-1/ICAM-1 (15 min). Data are shown as mean + SEM. Mann–Whitney test with P**** < 0.0001. Representative fluorescence microscopy images are shown (lower panel). The brightness of these images has been normalized to the highest value and is presented in false colors, ranging from dark blue (low fluorescence intensity) to bright yellow (high fluorescence intensity). The scale bar represents 10 µm

Fig 3: Characterization of integrin outside-in signaling nodes involved in VCAM-1/ICAM-1-induced mRNA expression of IL-1α and IL-1β. A Cartoon depicting the experimental design (Created in BioRender. Haage, R. (2025) https://BioRender.com/w21u020). B In 32D JAK2-VF cells, Il1a and Il1b mRNA expression is induced by the VLA4-VCAM-1 interaction (n = 4) but not by the β2-integrin-ICAM-1 interaction (n = 4). Specificity was tested by pre-incubation using 5 µg/ml anti-VLA4 or anti-β2-integrin antibody (n = 4). The stimulation of VCAM-1 and ICAM-1 in combination resulted in a further increase in adhesion-induced mRNA expression (orange; n = 3). C-D 32D JAK2-WT (blue) and 32D JAK2-VF (red) cells were stimulated with VCAM-1/ICAM-1 and various inhibitors were applied. The expression levels of (C) Il1a (n = 3) and (D) Il1b mRNA (n = 3) were measured upon inhibition of signaling molecules, including SRC (5 µM PP2), FAK (25 µM Y15), SYK (30 µM Piceatannol (Pic)), PI3K (50 nM Wortmannin (Wort)) and NFκB (50 µM JSH-23). E–F Clinical therapeutic drugs such as Ruxolitinib (2 µM RUX; orange) and Hydroxyurea (0.5 mM HU; blue) were tested for their effects on adhesion-induced (E) Il1a and (F) Il1b mRNA expression. To exclude the possibility of autocrine IL-1 signaling, IL1Ra was applied (dark grey). mRNA expression was measured by qPCR upon 3 h of stimulation and the resulting data are shown as fold change (2−ΔΔCT) as mean + SEM (n = 3–4). B Mixed-effects analysis (uncorrected Fisher´s LSD), C-D Friedman test (uncorrected Dunn´s test), E–F RM one-way ANOVA (uncorrected Fisher´s LSD) with P* < 0.05, P** < 0.01

Fig 4: Stimulation by VCAM-1/ICAM-1 induces protein expression of IL-1α/β in 32D JAK2-WT and 32D JAK2-VF cells. A-B VCAM-1/ICAM-1 stimulation increases intracellular (A) IL-1α and (B) IL-1β protein levels in 32D JAK2-WT (blue) and 32D JAK2-VF cells (red) in comparison to the BSA control (n = 4–6). After 6 h of stimulation, intracellular, mature IL-1α and IL-1β was measured by ELISA. The effects of PP2 (5 µM), Y15 (25 µM) and nigericin (5 µM) were investigated. 2-Way ANOVA with P* < 0.05, P** < 0.01, P*** < 0.001. C Following 24 h VCAM-1/ICAM-1 stimulation, significantly increased IL-1α surface expression was detected by flow cytometry in 32D JAK2-VF (red; n = 4) cells but not in 32D JAK2-WT cells (blue; n = 4). RM one-way ANOVA (Fisher´s LSD) with P** < 0.01, P*** < 0.001. D Upon VCAM-1/ICAM-1 stimulation (6 h), the presence of active caspase 1 was measured by FLICA (FAM-YVAD-FMK) using flow cytometry in 32D JAK2-WT cells (blue; n = 3) and 32D JAK2-VF (red; n = 4). As a positive control, cells were stimulated with LPS (100 ng/ml) and nigericin (5 µM). Ordinary one-way ANOVA with P* < 0.05, P** < 0.01, P*** < 0.001. E Intracellular and (F) secreted IL-1β protein levels of granulocytes isolated from Vav1-Cre x Jak2+/+ and Vav1-Cre x Jak2.VF/+ mice upon 6 h VCAM-1/ICAM-1 stimulation (n = 5) were measured. LPS (100 ng/ml) and ATP (3 mM) were utilized to investigate the effects of the inflammasome cascade on VCAM-1/ICAM-1 stimulation of IL-1α and IL-1β. They also served as positive controls. Data are shown as mean + SEM. Mixed-effects analysis (uncorrected Fisher´s LSD) with P* < 0.05, P** < 0.01, P*** < 0.001