Fig 2: IL-13 alone promoted phosphorylation of STAT6 and maturation and proliferation of SPEM cells. (A) Representative bright field images of Matrigel domes containing Meta1 gastroids treated with either rIL-13 or vehicle solution (BSA). Enlarged images from H&E staining show cellular distribution and height differences between the rIL–13-treated and the BSA-treated Meta1 gastroids. Quantitation of matched gastroid diameters showed significantly higher sizes in Meta1 gastroids treated with rIL-13 compared with control after 2 days of treatment. Scale bar: 1000 μm for brightfield and 100 μm for H&E. Mean ± SD (N = 3 independent experiments). ∗∗P < .01, ∗∗∗∗P < .0001. (B) Immunoblotting shows significant up-regulation of phosphorylated STAT6 in Meta1 gastroids treated with different concentrations of rIL-13 when compared with BSA. Quantitation showed rIL-13–dose-dependent increase. Mean ± SD (N = 3 independent experiments). ∗P < .05. (C) Immunofluorescence staining of Meta1 gastroids cross sections for SPEM-related markers, AQP5 and TFF2. Quantitation of AQP5 and TFF2 double positive cells demonstrated significant increase of proportion of mature SPEM cells in rIL-13–treated Meta1 gastroids. Yellow arrowheads point at double positive mature SPEM cells. Scale bar: 100 μm. Mean ± SD (N = 3 independent experiments). ∗∗∗P < .001. (D) Immunofluorescence staining of Meta1 gastroids cross sections for a proliferation marker, Ki67, and SPEM-related markers, MUC6 and CD44v9. Quantitation of MUC6 and CD44v9 double positive cells demonstrated significant increase of proportion of mature SPEM cells in rIL-13–treated Meta1 gastroids. Moreover, quantitation of MUC6, CD44v9, and Ki67 triple positive cells demonstrated significant increase of proportion of proliferative SPEM cells. Yellow arrowheads point at triple positive mature and proliferative SPEM cells. Scale bar: 100 μm. Mean ± SD (N = 3 independent experiments). ∗∗P < .01, ∗∗∗P < .001.

Fig 3: Mg2+ entry via TRPM7 channels facilitates the M2 phenotype.A Flow cytometer analysis on WT mice cells was performed to measure the levels of surface markers to demonstrate the efficacy of cell polarization. Data show a high CD206+ M2 marker and low CD80+ M1 marker to illustrate successful M2 polarization when naïve M0 cells are treated with IL-4 (20 ng/ml) for 24 h. Data shown are representative of three independent experiments with similar results. B Activated BMDM M1 cells cultured in 6 wells plate (2×106 cells/well) and treated with 1 mM and 5 mM Mg2+ and Ca2+ for 24 hr. The concentration of anti-inflammatory cytokine IL-1RA released in the media was performed by ELISA. Data shown are representative of three independent experiments with similar results. Bar graphs depict average ± SD for relative values, ***p ≤ 0.001 (Student’s t-test). C, D Flow cytometer analysis on M1 activated wild type mice BMDM treated with several Mg2+ concentrations (0 mM, 0.1 mM, 0.5 mM, 5 mM, 10 mM and 20 mM) for 24 hr to show how Mg2+ is relevant for macrophage polarization, increasing CD80 + M1 marker expression. Data shown are representative of three independent experiments with similar results. Bar graphs depict average ± SD for relative values, ***p ≤ 0.001 (Student’s t-test). E, F Activated BMDM M2 cells cultured in 6 wells plate (2×106 cells/well) and treated with 2APB (10 μM) plus 2APB + 20 mM Mg2+ and 2APB + 20 mM Ca2+; SKF (10 μM) plus SKF + 20 mM Mg2+ and SKF + 20 mM Ca2+ overnight. The concentration of anti-inflammatory cytokines (IL-10 and IL-1RA) released in the media was performed by ELISA. Data shown are representative of three independent experiments with similar results. Bar graphs depict average ± SD for relative values, NS = non-significant (Student’s t-test). G IV curves of TRPM7 current in M0, M2 cells and quantitation of current density at +100 mV (mean ± SD of 6 experiments) is shown in (H). Bath application 10 mM Mg2+, 20 mM Mg2+ induced inward current (holding at -120mV) are shown in (I). IV curves (J) and quantitation of current density at −120mV (K) in M0, M2 cells, as labeled in the figure. L Immunoblotting analysis showing the level of expression of TRPM7, pSTAT6, STAT6, and β-actin in wildtype mice M2 activated BMDM with increasing Mg2+ concentrations. Data shown are representative of three independent experiments with similar results. M Elisa quantification for VEGF in supernatants of M2 activated BMDM with 2APB treatment (10 µM overnight) and increasing Mg2+ concentrations. Data shown are representative of three independent experiments. Bar graphs depict average ±SD for relative values, ***p ≤ 0.001 (Student’s t-test). N, O Flow cytometer analysis on naïve BMDM (Mφ) with supplemented with 1 mM and 20 mM Mg2+ concentrations for 24 hr, showing the presence of M1 (CD 80) and M2 (CD 206) markers respectively. Bar graphs depict average ±SD for relative values, **p ≤ 0.005 (Student’s t-test).

Fig 4: JAK inhibitors suppressed IL-21 driven proliferative activities under anti-IgM challenge. Western blot analyses demonstrated that Ruxolitinib, Tofacitinib and Ritlecitinib inhibited (A) IL-4 triggered STAT6 phosphorylation and (B) IL-21 triggered STAT3 phosphorylation in a dose dependent manner. (C, D) WST-8 proliferative assays showed that all tested JAK inhibitors suppressed IL-4 and IL-21 rescues of anti-IgM arrested proliferation in Ramos cells. All groups shown in the figures were treated with 1 µg/ml anti-IgM for 48 hours. *p < 0.05 as compared to indicated DMSO control group by two-way ANOVA, N = 3.

Fig 5: Proanthocyanidins increased the phosphorylation of signal transducer and activator of transcription 6 (STAT6) in J774 macrophages. pSTAT6 protein levels were determined with Western blot and normalized to total STAT6. pSTAT6 expression in IL-4-treated cells was set as 100% (marked as black bar), and the other values are presented in proportion to that value. Actin was used as a loading control. Results are given as the mean + SEM, n = 4. One-way ANOVA with Dunnett’s post-test was used in the statistical analysis. Mean values significantly different from the control are marked with ** p < 0.01 and *** p < 0.001.